ZHANG, WENHUA. Structural Basis of the Biosynthesis of the tRNA N 6threonylcarbamoyladenosine. (Under the direction of H. van Tilbeurgh and codirection B. Collinet)

Most if not all tRNAs undergo chemical modifications during their maturation after the transcription. N 6 -threonylcarbamoyladenosine (t 6 A) is universally present at position 37 of tRNAs that recognize ANN-codons and it plays an essential role in translational fidelity through enhancing the codon-anticodon interaction. Recently, the tRNA t 6 A-modifying enzymes have been identified and characterized in bacteria, archaea and yeast. The biosynthesis of tRNA t 6 A proceeds in two main steps: first, the biosynthesis of an unstable intermediate threonylcarbamoyladenylate (TCA) by Sua5/YrdC family protein, using ATP, L-threonine, bicarbonate as substrates; second, the transfer of threonylcarbamoyl-moiety from TCA onto A 37 of cognate tRNAs by a set of other proteins that use Kae1/Qri7/YgjD family proteins as a catalytic component. Though the biosynthesis of tRNA t 6 A could be accomplished by Sua5 and Qri7 in yeast mitochondria, the t 6 A biosynthesis in archaea and yeast cytoplasm requires Sua5 and KEOPS protein complex, which consists of Kae1, Bud32, Cgi121, Pcc1 in archaea, and a fifth Gon7 in yeast. In bacteria, it requires YrdC, YgjD, YeaZ and YjeE, of which YeaZ and YjeE are not related to any KEOPS subunits. Presently, the molecular mechanism of Sua5/YrdC in catalyzing the TCA biosynthesis is not well understood; How the KEOPS subunits assembly and cooperatively transfer threonylcarbamoyl-moiety from TCA to tRNA is not known; The contribution of YeaZ and YjeE in t 6 A biosynthesis in bacteria still remains to be probed.

In this study, we report crystal structures of P. abyssi Sua5, S. cerevisiae Gon7/Pcc1 and Bud32/Cgi121 binary complexes, and E. coli YgjD-YeaZ heterodimer. Based on the information revealed by the crystal structures, advanced biochemical characterizations were carried out to validate the hypotheses. We confirm first that Sua5/YrdC is capable of catalyzing the TCA biosynthesis using substrates of ATP, L-threonine, and bicarbonate. The structure of P. abyssi Sua5 in complex with pyrophosphate provides a basis for its ATP-pyrophosphatase activity. Second, the structure of Gon7 reveals that it functions as a structural mimic of Pcc1 and therefore prevents the formation of Pcc1 homodimer, which mediates the formation of a dimer of tetrameric KEOPS from archaea. The structure of Bud32-Cgi121 in complex with ADP provides a basis in support of the dual kinase and ATPase activities of Bud32. We present a structural model of yeast KEOPS that exists as a heteropentamer. Third, we discovered that the weak intrinsic ATPase activity of YjeE is activated by YgjD-YeaZ heterodimer. YgjD, YeaZ and YjeE associate and form a ternary complex that is regulated by both the formation of YgjD-YeaZ heterodimer and the binding of ATP to YjeE. The model of YgjD-YeaZ-YjeE ternary complex provides structural insight into the essential role of YeaZ and YjeE in t 6 A biosynthesis in bacteria.

This work provides structural insights into understanding the biosynthesis of tRNA t 6 A that is essential and ubiquitous in all the three domains of life.

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II

ACKNOWLEDGMENT

It has been wholesome enjoyment to work with so many talented and passionate colleagues in the van Tilbeurgh's lab, in which people come and people go but people are connected and united in lifetime. I acknowledge the education and joys that I have recived from all former and current lab members.

INTRODUCTION 1 The central role of tRNA in genetic expression

A fundamental basis for understanding the genetic information transfer is the realization that genetic information is encoded in the DNA [START_REF] Avery | Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types : Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type Iii[END_REF]. The double helix DNA structure was discovered by James D. Watson and Francis H. C. Crick in 1953 and it revealed two universal modes of base pairing (G≡C and A=T) (Watson & Crick, 1953). In a simplified illustration of a possible copying mechanism, the genetic information encoded in the DNA is transcribed into a messenger RNA (mRNA) and then subsequently translated into amino acids, which are conjugated into proteins [START_REF] Crick | On protein synthesis[END_REF]. As simplified in the central dogma of molecular biology (Fig. 1A), the first fundamental stage of the genetic information transmission from the DNA sequence is the synthesis of a corresponding single stranded mRNA sequence (DNA → RNA) by transcription in the nucleus. This mRNA serves then as a template for the synthesis of protein (RNA → protein) by translation (Fig. 1B). It was soon realized that a strand mRNA is physically impossible to pair with a peptide due to a stereo-chemical non-complementarity between a nucleotide triplet and an amino acid [START_REF] Crick | On protein synthesis[END_REF]. Concomitantly, transfer RNA (tRNA) was proposed as the adaptor molecule that couples the recognition of the DNA codon and the incorporation of the specific amino acid during translation [START_REF] Hoagland | Intermediate reactions in protein biosynthesis[END_REF][START_REF] Zamecnik | Intermediate Reactions in Amino Acid Incorporation[END_REF]. Since a vast array of functions within the living systems are performed by proteins, any errors from DNA en route to protein could pose catastrophic dangers to the cell life. Thus, the translational fidelity ensures the consistency between DNA coding sequence and protein sequence. tRNA is at the core of accurate protein synthesis through their dual roles in both charging of the correct amino acids and specific recognition of codons located on mRNA (Fig. 1B) [START_REF] Jakubowski | Editing of errors in selection of amino acids for protein synthesis[END_REF].

The structure of tRNA

During translation tRNAs interact with both mRNA and ribosomes and this requires them to be chemically stable and structurally recognizable throughout the translation process. The high-resolution structures of tRNAs permitted the molecular understanding of the structures and functions of tRNAs in protein synthesis. Nascent tRNAs are composed of 4 types of nucleotides: adenine (A), guanine (G), uracil (U) and cytosine (C). All the nucleotides contain a base, a ribose sugar, and a phosphate (Fig. 2A). An important structural feature of RNA is the presence of a hydroxyl group at the position 2 of the ribose sugar. The presence of the 2'-OH group causes the helix to adopt A-form geometry and carries the potency to chemically attack the adjacent phosphodiester bond thereby cleaving the backbone [START_REF] Holley | Structure of a Ribonucleic Acid[END_REF][START_REF] Rich | Transfer RNA: molecular structure, sequence, and properties[END_REF]. All tRNAs have a canonical tertiary structure, which is essential for the recognition by tRNA-editing enzymes, aminoacyl tRNA synthetases (aaRSs) and ribosomes. Mature tRNA molecules typically contain 73-79 bases that fold into a 'cloverleaf'-like secondary structure, composed of five subdomains: acceptor stem, D-stem loop (DSL), anticodon stem loop (ASL), variable loop and the TΨC stem loop (TSL) [START_REF] Rich | The three-dimensional structure of transfer RNA[END_REF][START_REF] Robertus | Structure of yeast phenylalanine tRNA at 3 A resolution[END_REF]. The X-ray structure of yeast tRNA Phe revealed that the provides a physical possibility for tRNA to read the genetic information from the mRNA and to deliver the codon specified amino acid to the growing peptide on the ribosome [START_REF] Ramakrishnan | Ribosome structure and the mechanism of translation[END_REF]. Moreover, dynamic conformational flexibility has been observed for various tRNAs, suggesting that flexible regions between the DSL and TSL also play a role in discriminating the binding partners and in fine-tuning the function of tRNA [START_REF] Giege | Structure of transfer RNAs: similarity and variability[END_REF].

Fig. 2 The structures of tRNA. A. The primary structure of RNA in which the base, ribose and phosphate are highlighted in blue, red and green, respectively. B. The tertiary structure of yeast tRNA

Phe (PDB ID: 4TNA) in which the subdomains are labeled.

The maturation of tRNA

The correct translation of mRNA during protein synthesis is directly correlated with tRNA aminoacylation and decoding efficiency. tRNA is transcribed as a precursor that has to be edited during a process called "tRNA maturation". This process consists of removal of oligonucleotides and introduction of a number of chemical modifications. Thus, the nascent tRNAs undergo several maturation steps necessary for adopting its tertiary structure which will be recognized during the protein synthesis process. By increasing structural diversity and functional adaptability, modified nucleosides of tRNA play an important role in gene expression.

Fig. 3 The maturation and functional implication of tRNA in cell life. The tRNA transcripts undergo several processing and editing steps, including removal of introns, addition of CAA at the 3' terminus as well as a plethora of chemical modifications. Chemical modifications of tRNA substrates can be introduced in the nucleus, cytoplasm, or in organelles. (Abbreviations: RTD, rapid tRNA decay pathway; TRAMP, Trf4/Air2/Mtr4p polyadenylation complex, AARSs, aminoacyl tRNA synthetases, EF-Tu, elongation factor thermo unstable). The figure was taken from [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF].

The processing of the nascent tRNAs

Transcribed tRNA molecules possess extra nucleotides at both the 5'-and 3'-terminus. The 5' leader sequence and 3' trailer sequence are removed progressively by essential ribonucleoprotein RNase P and endonuclease RNase Z, respectively [START_REF] Chamberlain | Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP[END_REF][START_REF] Takaku | A candidate prostate cancer susceptibility gene encodes tRNA 3' processing endoribonuclease[END_REF]. Introns are sometimes present in tRNA genes and they must be spliced before tRNAs can function in protein synthesis. The splicing of introns from eukaryotic tRNA precursors in nucleus involves both cleavage and ligation. The splicing endonuclease recognizes the introns and cleaves at both ends of the tRNA precursor, followed by ATP-dependent bond synthesis by RNA ligase [START_REF] Abelson | tRNA splicing[END_REF][START_REF] Trotta | The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases[END_REF]. One of the most essential tRNA editing processes is the addition of a CCA triplet at the 3' terminus, on which the tRNA is subsequently used to charge the correct amino acid by one specific tRNA synthe-tase. Despite the 3' terminal CCA is encoded in the gene in prokaryotes, it still requires the tRNA nucleotidyl transferase to repair the 3' terminus when damaged [START_REF] Zhu | tRNA nucleotidyltransferase is not essential for Escherichia coli viability[END_REF]. In contrast, the CCA at 3' terminus of tRNA is not encoded in the gene in eukaryotes and therefore, the maturation of tRNA from eukaryotes requires the addition of the CCA triplet by nucleotidyl transferase [START_REF] Aebi | Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae[END_REF]. During the maturation of tRNA precursor, a vast number of modifications are progressively introduced, a process that takes place in both nucleus (eukaryotes) and cytoplasm (Fig. 3) [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF]. Moreover, some modifications necessitate the retrograde import of the premature tRNA from cytoplasm to the nucleus [START_REF] Shaheen | Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae[END_REF].

Fig. 4 Overview of the modified nucleosides of tRNA. A. The occurrence of chemically modified nucleosides in tRNAs from the three domains of life [START_REF] Jackman | Transfer RNA modifications: nature's combinatorial chemistry playground[END_REF]. The shaded areas represent the co-occurrence of modifications and brackets denote modifications that take place in mitochondria. B. The presence of the modifications in tRNAs from yeast [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF]. The two figures were taken from [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF][START_REF] Jackman | Transfer RNA modifications: nature's combinatorial chemistry playground[END_REF].

The identity of tRNA by modification

Chemically modified nucleosides are found in all types of nucleic acids, with higher abundance in noncoding RNAs. All mature tRNAs display varying degrees of chemical modifications in bacteria, archaea and eukarya (Fig. 4) [START_REF] Grosjean | Posttranscriptionally modified nucleosides in transfer RNA: their locations and frequencies[END_REF][START_REF] Jackman | Transfer RNA modifications: nature's combinatorial chemistry playground[END_REF][START_REF] Phizicky | tRNA biology charges to the front[END_REF].

Most modifications occurring at different positions of tRNAs contribute to the idiosyncratic features that are of functional and structural significance. The degree, the extent and the pattern of the modifications depends on physiological conditions, tRNA species and organisms [START_REF] Novoa | Speeding with control: codon usage, tRNAs, and ribosomes[END_REF]. While some modifications alter the structure of tRNA and ensure the Table 1 The enzymes that are required in the modifications present at positions 34 (blue part) and 37 (red part) of tRNAs. The table was adapted from [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF].

during the protein translation on ribosome [START_REF] Phizicky | tRNA biology charges to the front[END_REF][START_REF] Vendeix | Anticodon domain modifications contribute order to tRNA for ribosome-mediated codon binding[END_REF]. While most modifications are only observed in specific organism(s), some sets of essential modifications are universally present in tRNAs from all three domains of life. The complexity of the chemical modification varies at different positions (Fig. 4A). Some modifications require single enzymes, while others invoke the successive actions of more than one enzyme (Table 1) [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF]. The modifications at the backbone of tRNAs play a crucial role in conferring the key recognition features to tRNAs upon interaction with tRNA synthetases or a number of tRNA-modifying enzymes. Single-nucleoside substitutions of tRNAs have been reported in several systems. A number of tRNAs undergo a conversion of adenosine to inosine (A to I) at position 57 by deaminases, a process also involving an a priori methylation of adenosine by methylases [START_REF] Keller | Editing of messenger RNA precursors and of tRNAs by adenosine to inosine conversion[END_REF]. Thus, methylinosine (m 1 I) at position 57 is eventually formed in the TΨC loop of many tRNAs.

The C to U substitution at position 28 of tRNAs from plant mitochondria occurs at a position where pseudourine (Ψ) is found at the anticodon stem loop [START_REF] Ferreira-Cerca | ATPase-dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit[END_REF]. Thus it was hypothesized that the conversion of C to U at position 28 is required for isomerization of the U to Ψ. Of all the chemical distinct modifications that play a direct role in the decoding process, most are universally introduced at the two hyper-modified positions: the wobble position 34 and the position 37, 3'-adjacent to the anticodon (Table 1). For example, Ψ and m 5 C at position 34 and m 1 G and t 6 A at position 37 are required to modulate the decoding capability of tRNAs [START_REF] Yarian | Accurate translation of the genetic code depends on tRNA modified nucleosides[END_REF].

The aminoacylation of tRNA

The overall fidelity of protein synthesis is mostly dependent on aminoacylation of tRNAs and the subsequent codon-anticodon recognition. The synthesis of aminoacyl-tRNAs is accomplished by aminoacyl-tRNA synthatases (aaRSs) that catalyze the esterification of a specific amino acid to one of all its cognate tRNAs. Since the errors in the amino acid charging of the tRNAs will unlikely be detected or corrected by proofreading during the subsequent ribosomal incorporation steps. tRNA-synthetases must therefore recognize very precisely both the amino acid and its corresponding tRNA [START_REF] Ibba | Aminoacyl-tRNA synthesis[END_REF]. The 20 aminoacyl-tRNA synthetases are grouped into class I and class II based on structural and sequence similarities [START_REF] Eriani | Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs[END_REF]. The enzymes from the two classes have entirely different structures [START_REF] Cusack | Eleven down and nine to go[END_REF]. For instance, the ATP-binding domain of class I is a Rossmann fold with parallel β-strands whereas the corresponding domain in class II is formed by anti-parallel βstrands. While class I aaRSs charge amino acids to the tRNA on the 2'-OH moiety, class II aaRSs charge on the 3'-OH (Fig. 5A). [START_REF] Ibba | Aminoacyl-tRNA synthesis[END_REF].

A two-step mechanism has been proposed for the amino acid charging onto its cognate tRNA: first, an aaRS catalyzes the formation of the reactive aa-AMP when correct amino acid and ATP are bound in the catalytic center of aaRS; secondly, the aaRS catalyzes the transfer of amino acid from aa-AMP to 2'-or 3'-OH group of the terminal adenosine of the correct tRNA that is bound adjacently to the aa-AMP in the active site (Fig. 5B). The aminoacyla-

A B

tion reaction also produces one molecule of pyrophosphate and one molecule of AMP [START_REF] Ibba | Aminoacyl-tRNA synthesis[END_REF]. While the cellular abundance of amino acids plays a major role in the binding of amino acids to cognate aaRSa, the specific recognition of amino acids is achieved by specific contacts and hydrolysis of the non-cognate aa-tRNAs that involves the editing and proofreading activities of aaRSs, respectively [START_REF] Jakubowski | Editing of errors in selection of amino acids for protein synthesis[END_REF]. The identification of the correct tRNA by an aaRS is mainly achieved by its specific interaction with the discriminator base at position 73 of tRNA, the acceptor stem and the anticodon [START_REF] Hou | Discriminating among the discriminator bases of tRNAs[END_REF][START_REF] Moras | Structural aspects and evolutionary implications of the recognition between tRNAs and aminoacyl-tRNA synthetases[END_REF][START_REF] Saks | The transfer RNA identity problem: a search for rules[END_REF]. Nonetheless, the interactions with other regions of tRNA and the disordered regions of aaRSs are mutually important for discrimination of the binding of cognate tRNAs [START_REF] Naganuma | The selective tRNA aminoacylation mechanism based on a single G*U pair[END_REF][START_REF] Seif | RNA-protein mutually induced fit: structure of Escherichia coli isopentenyl-tRNA transferase in complex with tRNA(Phe)[END_REF][START_REF] Yang | Structural disorder in expanding the functionome of aminoacyl-tRNA synthetases[END_REF]. A plethora of chemical modifications of tRNA are involved in the tRNA aminoacylation. The functional role of these modifications present on tRNAs may contribute the faithful aminoacylation by enhancing the tertiary structure or/and inducing a favorable local conformation of tRNA [START_REF] Jackman | Transfer RNA modifications: nature's combinatorial chemistry playground[END_REF][START_REF] Naganuma | The selective tRNA aminoacylation mechanism based on a single G*U pair[END_REF].

However, the mechanisms accounting for the specific modifications remain to be investigated as the unmodified tRNA transcripts are capable for aminoacylation with cognate amino acids in some cases [START_REF] Zeevi | Aminoacylation and nucleoside modification of in vitro synthesised transfer RNA[END_REF].

tRNA-modifying enzymes aaRSs Ribosome

Fig. 6 The schematic modes of the interactions between tRNA and tRNA-modifying enzymes, and aaRSs, and ribosomes.

Recognition of tRNAs in the cell

tRNAs in their life cycle contact a large number of different proteins throughout its maturation, aminoacylation and protein synthesis. Thus, a range of different physical interactions occurs between tRNAs (or tRNA pre-cursors) and a large number of binding partners, including tRNA processing and modifying enzymes, aaRSs, ribosomes and mRNA (Fig. 6). The mutual selection of tRNAs and their binding partner(s) is mediated by both nonspecific interactions guided by the structural architecture and the electrostatic surface and specific interactions generated by induced fit and stereo complementarity. tRNA identity and structural features of the tRNA-binding partners are in general the major determinants of the specific interactions.

The recognition of tRNAs by tRNA-modifying enzymes

tRNAs inside the cell likely exist in several alternative conformations, thus making the recognition by tRNA-modifying enzymes much more selective [START_REF] Giege | Structure of transfer RNAs: similarity and variability[END_REF]. tRNA-modifying enzymes are most frequently characterized by an α/β fold that is frequently found in most nucleic acid-binding proteins. Moreover, they also tend to exhibit positively charged surface patches in favor of binding the negatively charged tRNAs. All the tRNA-modifying enzymes contain at least one catalytic domain and one binding domain. For some tRNA modifying enzymes, the interaction with tRNAs involves rigid fit.

For example, the crystal structures of MnmA-tRNA complex reveal that the 2thiouridine synthetase MnmA binds the substrate tRNA in a predominantly rigid manner. The sequential interactions between MnmA and tRNA only involve the limited conformational change of the anticodon stem loop of tRNA.

The selectivity, however, is determined by the base-flipping mechanism, by which the U 34 is flipped out and inserted into the active site of MnmA [START_REF] Guelorget | Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes[END_REF]. By contrast, the tRNA adenosine deaminase TadA binds the tRNA Phe in a more sophisticated manner in which pronounced conformational change of anticodon stem loop was observed [START_REF] Luo | Transition state structure of E. coli tRNA-specific adenosine deaminase[END_REF]. Much of the energetic driving force for remodeling the tRNA tertiary structure is produced by the favorable interactions between the interacting loop of tRNA and TadA surface. The stabilization of TadA with tRNA is further reinforced by a direct minor groove interaction with C 32 and A 38 . TadA recognizes its substrate via hydrogen bonding between C 35 and G 36 [START_REF] Losey | Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA[END_REF]. More interestingly, the pseudourine synthase TruA recognizes and modifies a wide range of nucleosides (38)(39)(40) on the anticodon stem loop [START_REF] Dong | Crystal structure of tRNA pseudouridine synthase TruA from Thermus thermophilus HB8[END_REF]. The crystal structures of TruA-tRNA complex reveal that the interaction requires dimer of TruA and the two molecules of tRNA interact with both monomers. While one monomer of TruA dimer interacts with the anticodon stem loop of the tRNA, the other monomer interacts with Dstem loop and TΨC loop of the same tRNA [START_REF] Hur | How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA[END_REF]. The dynamic motion of the anticodon stem loop allows the potential modification bases access to the active site of TruA. The structural snapshots show that the intrinsic conformational plasticity of the anticodon stem loop is the determinant of the multi-site specificity of TruA [START_REF] Guelorget | Mechanism-based strategies for trapping and crystallizing complexes of RNA-modifying enzymes[END_REF]. In general there are three different modes on the initial binding: 1) one enzyme is required to bind and catalyze the methylation; 2) two enzymes are required for efficient binding and subsequent catalysis and 3), one or more enzymes are needed forming a complex for the binding and catalysis (Fig. 6).

The recognition of tRNAs by aaRSs

The specific recognition of tRNAs is an essential event for interactions between aaRSs and tRNAs, which discriminate the potential promiscuous binding of tRNAs to aaRS-like proteins such as TagD family proteins and ATP sulfurlases [START_REF] Bork | The cytidylyltransferase superfamily: identification of the nucleotide-binding site and fold prediction[END_REF][START_REF] Mougous | Molecular basis for G protein control of the prokaryotic ATP sulfurylase[END_REF]. The tertiary structure, the local rearrangement of the backbone and the specific hydrogen-bonded interaction are the major contributions of tRNAs in their interaction with aaRSs [START_REF] Qin | Cocrystal Structures of Glycyl-tRNA Synthetase in Complex with tRNA Suggest Multiple Conformational States in Glycylation[END_REF]. Of utmost importance is the interaction between anticodon of tRNAs and anticodon-binding domain of aaRSs [START_REF] Moras | Structural aspects and evolutionary implications of the recognition between tRNAs and aminoacyl-tRNA synthetases[END_REF]. More mechanistically, a recent report demonstrated that a single wobble pair, G3≡U70, of tRNA Ala is also a major determinant to realize the specificity between tRNA Ala and alanyl-tRNAsynthetase (alaRS) [START_REF] Naganuma | The selective tRNA aminoacylation mechanism based on a single G*U pair[END_REF]. In addition, the modified nucleosides of tRNAs also contribute a higher affinity and specificity.

In contrast to structurally homogeneous tRNAs, large structural variations among aaRSs contribute a major determinants throughout the mutual recognition between tRNAs and aaRSs [START_REF] Cavarelli | Recognition of tRNAs by aminoacyl-tRNA synthetases[END_REF]. Class I aaRSs bind the acceptor of tRNAs at the minor groove with the variable loop exposed to the solvent, whereas Class II aaRSs bind at the major groove with the variable loop occluded from the solvent. In addition to the different surface contacts between tRNA and two classes of aaRSs, the conformations of the CCA triplet at the 3'-terminus are different, i.e, the CCA is bent to be accommodat-ed into the active site of class I aaRSs compared to that in class II aaRSs [START_REF] Ibba | Aminoacyl-tRNA synthesis[END_REF]. Other domains of aaRS also contribute to the specific recognition of tRNA by providing contacts with regions of the tRNA, i.e, the Cterminal α-helical domain.

The recognition of tRNAs by ribosomes

The selection of tRNAs by the ribosome is central to translational fidelity, which involves both favorable stereo complementarity and faithful codonanticodon pairing. During initiation of the translation mRNA and an initiator tRNA are bound between the cleft of the large and small subunits of the ribosome, forming the initiation complex. The subunit arrangement fits the Lshaped structure of tRNA with the anticodon at one end and the charged amino acid at the opposite end. During translation, tRNAs bind sequentially at three sites on the ribosome: the A-, P-and E-sites, to which the tRNAs are bound in transitional states called hybrid states [START_REF] Moore | How should we think about the ribosome?[END_REF][START_REF] Wilson | The structure and function of the eukaryotic ribosome[END_REF]. The binding of tRNA to the ribosome involves both the rRNA and ribosomal proteins [START_REF] Noller | Translocation of tRNA during protein synthesis[END_REF]. At the A-site, tRNA is accommodated and stabilized by its anticodon paired to the codon on the small subunit and by the acceptor interacting with cognate amino acids in the peptidyl transfer center on the large subunit. The rRNAs in the small subunit at the decoding center are also directly involved in the recognition of tRNA [START_REF] Li | Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM[END_REF]. The tRNA exhibits more conformational changes in the P-site where the tRNA has numerous contacts with the ribosome. Furthermore, the CCA triplet of the tRNA stacks with the G 2252 and G 2251 bases on the ribosome and the tRNA acceptor makes specific interactions with surrounding ribosomal proteins. The deacylated tRNA moves out of the ribosome from the E site, the end of tRNA tunnel of the subunit interface. The anticodon stem loop of tRNA at the P site is positioned by its interaction with rRNA and several ribosomal proteins [START_REF] Spahn | Structure of the 80S ribosome from Saccharomyces cerevisiae--tRNAribosome and subunit-subunit interactions[END_REF][START_REF] Yusupov | Crystal structure of the ribosome at 5.5 A resolution[END_REF].

The chemistry, biology and biosynthesis of tRNA t 6 A

The N 6 -threonylcarbamoyladenosine (t 6 A) was first isolated from E. coli tRNAs that recognize the ANN-codons in 1969 [START_REF] Ishikura | The presence of N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine in serine, methionine and lysine transfer RNA's from Escherichia coli[END_REF]. Later on, the substrates for biosynthesis were found to be tRNA, ATP, L-threonine and bicarbonate [START_REF] Elkins | The enzymatic synthesis of N-(purin-6ylcarbamoyl)threonine, an anticodon-adjacent base in transfer ribonucleic acid[END_REF][START_REF] Korner | N-(purin-6-ylcarbamoyl)threonine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli[END_REF]. The t 6 A plays a pivotal role in maintaining translational fidelity [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF][START_REF] Fvt | The role of modifications in codon discrimination by tRNA(Lys)UUU[END_REF][START_REF] Parthasarathy | Conformation and possible role of hypermodified nucleosides adjacent to 3'-end of anticodon in tRNA: N-(purin-6-ylcarbamoyl)-L-threonine riboside[END_REF]Weissenbach & Grosjean, 1981a). However, the enzyme(s) responsible for the biosynthesis of tRNA t 6 A had remained a mystery for almost four decades. Only recently, the complete enzymes responsible for the in vitro biosynthesis of tRNA t 6 A have been discovered in bacteria, archaea and yeast [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Fig. 7 The t 6 A of tRNA. A. t 6 A is present at position 37 of tRNAs that decode ANN-codons. B. the chemical structure of t 6 A. C. the 3D structure of t 6 A (colored in red) in crystal structure of T. thermophilus tRNA LysUUU in complex with mRNA (PDB ID: 1XMO).

The characterization of tRNA t 6 A

Chemical characterization of t 6 A has gained particular interest for being one of the very few nucleosides onto which an amino acid is introduced. The biological importance has been intensively probed after t 6 A had been assigned 3' adjacent to the anticodon of tRNA (Fig. 7). The co-occurrence of the lack of tRNA t 6 A and defective protein translation suggested that t 6 A is principally related to translational fidelity.

The discovery of tRNA t 6 A and chemical characterization

The N-(purin-6-ylcarbamoyl)-l-threonine riboside (Fig. 7), the original chemical description of t 6 A, was first isolated from digested nucleosides of tRNAs from bacteria, yeast and human [START_REF] Chheda | Isolation and characterization of a novel nucleoside, N-[(9beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]threonine, from human urine[END_REF][START_REF] Chheda | Aminoacyl nucleosides. VI. Isolation and preliminary characterization of threonyladenine derivatives from transfer ribonucleic acid[END_REF][START_REF] Ishikura | The presence of N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine in serine, methionine and lysine transfer RNA's from Escherichia coli[END_REF][START_REF] Schweizer | Aminoacyl nucleosides. VII. N-(Purin-6-ylcarbamoyl)threonine. A new component of transfer ribonucleic acid[END_REF]. The presence of t 6 A was exclusively

A B C
observed in ANN-decoding tRNAs such as tRNA Ser , tRNA Met , tRNA Lys and tRNA Asn from E. coli [START_REF] Ishikura | The presence of N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine in serine, methionine and lysine transfer RNA's from Escherichia coli[END_REF][START_REF] Kimura-Harada | The presence of N-[9-(c-Dribofuranosyl)purin-6-ylcarbamoyl] threonine in isoleucine, threonine and asparagine tRNAs from Escherichia coli[END_REF].

Sequence determination of the yeast tRNA Ile revealed that the t 6 A was present 3' adjacent to anticodon of tRNAs [START_REF] Takemura | The primary structure of isoleucine transfer ribonucleic acid from Torulopsis utilis. Complete digestion with ribonucleases and construction of model of its secondary structure[END_REF]. The chemical composition of t 6 A includes an adenosine and a N-carbamoylthreonine that is composed of a carboxylic group and a L-threonine group [START_REF] Schweizer | Aminoacyl nucleosides. VII. N-(Purin-6-ylcarbamoyl)threonine. A new component of transfer ribonucleic acid[END_REF]. The in vivo incorporation of the L-[U- 14 C]threonine into the t 6 A of rat liver and E. coli demonstrated that the L-threonine is a precursor for the Ncarbamoylthreonine-moiety [START_REF] Chheda | Biosynthesis of N-(purin-6-ylcarbamoyl)-L-threonine riboside. Incorporation of L-threonine in vivo into modified nucleoside of transfer ribonucleic acid[END_REF]. Carbamoyl phosphate was anticipated at the very beginning as the donor of the carbonyl group for the Ncarbamoylthreonine-moiety [START_REF] Chheda | Biosynthesis of N-(purin-6-ylcarbamoyl)-L-threonine riboside. Incorporation of L-threonine in vivo into modified nucleoside of transfer ribonucleic acid[END_REF][START_REF] Powers | Biosynthesis and specific labeling of N-(purin-6-ylcarbamoyl)threonine of Escherichia coli transfer RNA[END_REF]. Later on, the biosynthesis of the tRNA t 6 A was reconstituted by partially-purified enzymes (cell lysate) only in presence of t 6 A-deficient tRNA, ATP, L-threonine and bicarbonate, demonstrating that, however, bicarbonate is the bona fide donor of carbonyl group [START_REF] Elkins | The enzymatic synthesis of N-(purin-6ylcarbamoyl)threonine, an anticodon-adjacent base in transfer ribonucleic acid[END_REF]. This is further corroborated by the substitution of bicarbonate by CO 2 under certain circumstances [START_REF] Elkins | The enzymatic synthesis of N-(purin-6ylcarbamoyl)threonine, an anticodon-adjacent base in transfer ribonucleic acid[END_REF][START_REF] Korner | N-(purin-6-ylcarbamoyl)threonine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

Apart from several isopentenyl-adenosine derivatives such as i 6 A, ms 2 i 6 A, and ms 2 io 6 A as shown in Fig. 8, the modified nucleosides of ms 2 t 6 A, m 6 t 6 A and ct 6 A are derivatives of t 6 A at position 37 in certain types of tRNAs (Fig. 4A) [START_REF] Cantara | The RNA Modification Database, RNAMDB: 2011 update[END_REF][START_REF] Kimura | Discovery of the beta-barrel-type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs[END_REF][START_REF] Soll | Enzymatic modification of transfer RNA[END_REF]. The ms 2 t 6 A modification present in several bacterial and eukaryotic tRNAs is essential for efficient and accurate decoding [START_REF] Arragain | Identification of eukaryotic and prokaryotic methylthiotransferase for biosynthesis of 2-methylthio-N6threonylcarbamoyladenosine in tRNA[END_REF]. The biosynthesis of ms 2 t 6 A requires sulfur insertion with methylation at C 2 of t 6 A by the methylthiotransferase MtaB in B. subtilis and Cdk11 in human, which belong to the methylthiotransferase (MTTase) superfamily [START_REF] Durant | Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A[END_REF]. In E. coli, m 6 t 6 A is only present in tRNAs Thr1/3(GGU) that decode ACY codons [START_REF] Komine | Nucleotide sequence of tRNA(Thr1) of Escherichia coli and of the gene (thrV) that encodes it[END_REF]. It was shown that the N 6 -methyl group of m 6 t 6 A, which originates from methionine, reinforces the reading of the cognate codon ACC from the thr leader [START_REF] Qian | The methyl group of the N6-methyl-N6threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA[END_REF]. Very recently, TrmO from bacteria and human was identified as the methyltransferase responsible for N 6 -methyl group of m 6 t 6 A, using S-adenosyl-L-methionine as a methyl donor [START_REF] Kimura | Discovery of the beta-barrel-type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs[END_REF]. During early characterization of the chemical synthesis of t 6 A, the existence of the cyclic t 6 A (ct 6 A) was observed as an intermediate of t 6 A due to the hydrolytic treatment [START_REF] Schweizer | Aminoacyl nucleosides. VII. N-(Purin-6-ylcarbamoyl)threonine. A new component of transfer ribonucleic acid[END_REF]. The in vivo existence of ct 6 A was recently identified in some tRNAs, i.e. tRNA Thr2(CGU) and tRNA Thr4(UGU) , from bacteria to plants and has been directly required for promotion of decoding efficiency [START_REF] Miyauchi | A cyclic form of N(6)threonylcarbamoyladenosine as a widely distributed tRNA hypermodification[END_REF]. In addition, TcdA was identified as the enzyme responsible for dehydration of t 6 A to form ct 6 A in E. coli. Tcd1 and Tcd2 that are yeast homologs of TcdA were shown to be required for formation of ct 6 A [START_REF] Miyauchi | A cyclic form of N(6)threonylcarbamoyladenosine as a widely distributed tRNA hypermodification[END_REF]. However, ct 6 A was not identified in eukaryotic mitochondrion [START_REF] Suzuki | A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs[END_REF].

Fig. 8 Schematic representation of N 6 -threonylcarbamoyladenosine (t 6 A) structure. A few other chemically distinct modifications on adenosine at position 37 of tRNAs are also shown: ct 6 A, m 6 t 6 A, ms 2 t 6 A, hnt 6 A, g 6 A. The figure is adapted from [START_REF] Matuszewski | Stability studies on the newly discovered cyclic form of tRNA N(6)-threonylcarbamoyladenosine (ct(6)A)[END_REF][START_REF] Morin | Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t6A) in tRNA: an in vivo study with Xenopus laevis oocytes[END_REF]. For the common chemical names and symbols, please refer to appendix 1.

The essential role of t 6 A in translational fidelity

The biological importance of t 6 A for the structure and function of tRNA is related to its large chemical group of L-threonine and its occurrence 3'-adjacent to the anticodon (Fig. 7). Early characterization of t 6 A suggested it might contribute to the stability for the tRNA-mRNA-ribosome complex by means of electrostatic interaction with the basic ribosomal proteins [START_REF] Schweizer | Aminoacyl nucleosides. VII. N-(Purin-6-ylcarbamoyl)threonine. A new component of transfer ribonucleic acid[END_REF][START_REF] Takemura | The primary structure of isoleucine transfer ribonucleic acid from Torulopsis utilis. Complete digestion with ribonucleases and construction of model of its secondary structure[END_REF]. The X-ray crystal structures of t 6 A revealed that threonyl-moiety is cis to N 1 of the adenine, enabling HN 11 to hydrogen-bond with N 1 (Fig. 8) [START_REF] Fvt | The role of modifications in codon discrimination by tRNA(Lys)UUU[END_REF][START_REF] Parthasarathy | Conformation and possible role of hypermodified nucleosides adjacent to 3'-end of anticodon in tRNA: N-(purin-6-ylcarbamoyl)-L-threonine riboside[END_REF]. This conformation disables t 6 A in taking part in Watson-Crick base pairing, suggesting a structural role of t 6 A in promoting a single stranded conformation for the anti-codon stem loop [START_REF] Parthasarathy | Conformation and possible role of hypermodified nucleosides adjacent to 3'-end of anticodon in tRNA: N-(purin-6-ylcarbamoyl)-L-threonine riboside[END_REF]. Thermodynamic and kinetic analyses demonstrated that t 6 A confers significant stabilizing effect on the U=A pairing 5' adjacent to t 6 A (Weissenbach & Grosjean, 1981b). Further biochemical studies of the effect of t 6 A modification on tRNAs demonstrated that t 6 A confers stabilizing effect to the tertiary structure of tRNA and mediates the interactions among anticodon, codon and ribosome during protein translation [START_REF] Motorin | tRNA stabilization by modified nucleotides[END_REF][START_REF] Vendeix | Anticodon domain modifications contribute order to tRNA for ribosome-mediated codon binding[END_REF]. The crystal structure of the T. thermophilus tRNA Lys anticodon stem loop (ALS) bound to the 30S ribosomal subunit in which a codon-containing mRNA fragment is also bound revealed that the formation of an intra-loop between U 33 and A 37 on human tRNA Lys is disrupted by the presence of t 6 A. The extended ring structure of t 6 A 37 enables cross-strand base-stacking of this modified nucleoside in the anticodon loop with A 38 of tRNA and with A1, the base in the first position of the codon [START_REF] Fvt | The role of modifications in codon discrimination by tRNA(Lys)UUU[END_REF]. As expected, the GFP reporter system demonstrated that the deficiency of t 6 A in tRNA leads to aberrant initiation at GUG codons, massive frameshift, inability to recognize the stop codons during translation as well as a range of pleiotropic phenotypes [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF][START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Miyauchi | A cyclic form of N(6)threonylcarbamoyladenosine as a widely distributed tRNA hypermodification[END_REF]. Nonetheless, t 6 A was further implicated in a number of other important biological processes. For instance, t 6 A is a determinant of isoleucine identity that is essential for lysidine synthetase to catalyze the formation of K 2 C 34 in the CAU-containing precursor tRNA Ile [START_REF] Grosjean | Enzymatic conversion of cytidine to lysidine in anticodon of bacterial isoleucyl-tRNA--an alternative way of RNA editing[END_REF]. And, the requirement of t 6 A for cleavage of tRNA Lys 5' to the wobble base suggested that the t 6 A is the determinant for cleavage of the tRNA anticodon by PrrC in E. coli [START_REF] Jiang | Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs[END_REF].

The in vitro enzymatic reconstitution of tRNA t 6 A

Comparative genomics and biochemical experiments documented the roles of Sua5/YrdC family and Kae1/Qri7/YgjD family in tRNA t 6 A modification [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF][START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF]. Sua5 is consisted of Nterminal YrdC domain and C-terminal Sua5 domain. YrdC domain is universally present in all the sequence genome and exists as an independent unit in some genomes like E. coli (Fig. 9). It was soon realized that neither Kae1 and Sua5 from yeast nor YgjD and YrdC from bacteria are sufficient for the in vitro biosynthesis of tRNA t 6 A, suggesting other accessory enzymes are essentially involved [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF]. Kae1 is the most conserved component of the KEOPS protein complex that also comprises Bud32, Cgi121 and Pcc1 in archaea, and a fifth fungi-specific protein Gon7 in yeast (Fig. 10A). It was later on demonstrated that KEOPS and Sua5 are necessary and sufficient for the in vitro biosynthesis of tRNA t 6 A in yeast cytoplasm and archaea [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]Kisseleva-Romanova et al, 2006b;[START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. In bacteria, it was concomitantly discovered that YeaZ and YjeE are needed in conjunction with YgjD and YrdC [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF]. Very recently, yeast mitochondrial Qri7 and Sua5 have been demonstrated to constitute the minimal system for tRNA t 6 A biosynthesis [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The biosynthesis of tRNA t 6 A was proposed to proceed in two steps: first, the Sua5/YrdC utilizes ATP, L-threonine and bicarbonate to synthesize an intermediate threonylcarbamoyladenylate (TCA), of which the threonylcarbamoyl-moiety is transferred by other protein components onto A 37 of substrate tRNA in the second step (Fig. 10B) [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Fig. 10 The overview of tRNA t 6 A biosynthesis. A. The enzymes required for the in vitro biosynthesis of t 6 A in each respective system. B. The proposed two-step reaction scheme in which proteins ① are responsible for the biosynthesis of TCA and proteins ② are responsible for transfer of threonylcarbamoyladenylate onto A 37 of substrate tRNA.

The t 6 A biosynthesis by KEOPS and Sua5 in yeast cytoplasm

The deletions of BUD32, PCC1 and GON7 abolished the in vivo formation of tRNA t 6 A in S. cerevisiae, manifesting that other subunits of KEOPS are required for biosynthesis of t 6 A, apart from the previously discovered Kae1 and Sua5 [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. While the mutations on the conserved metal-binding motif of Kae1 abolished the in vivo formation of t 6 A, the mutation on putative active site of Kinase Bud32 leads to substantially decreased level of t 6 A, indicating that Kae1 is more crucial for the in vivo for-A B mation of tRNA t 6 A [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. Subsequently, the in vitro biosynthesis of tRNA t 6 A was accomplished by KEOPS in conjunction of Sua5, using the tRNA, ATP, L-threonine and bicarbonate as substrates [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Of particular interest, the heterologous complementation of S. cerevisiae Sua5 by E. coli YrdC or P. abyssi Sua5 in the in vitro t 6 A biosynthesis demonstrated for the first that the Sua5/YrdC proteins could function without specific interaction with KEOPS. It was also shown that KEOPS and Sua5 from yeast are still capable of modifying the substrate tRNAs from bacteria, indicating an universal and conserved catalytic center present in these t 6 Amodifying enzymes in from all three domains of life [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

Kae1 is the first most conserved component of KEOPS and its orthologs are present in all the sequenced genomes of the three domains of life. Structural analysis showed that Kae1 possess an HSP70-actin fold and a conserved metal-binding motif of HxxxH-D, which are also characteristic features of all Kae1/Qri7/YgjD family proteins (Hecker et al, 2007b). The binding of ATP to P. abyssi Kae1 and M. jannaschii Kae1 from archaea has been determined and also, very weak intrinsic ATPase activity has been observed for P. abyssi Kae1 (Hecker et al, 2007b) and S. cerevisiae Kae1 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

Though the mutations on metal-binding motif of Kae1 do not affect the ATPase atctivity of S. cerevisiae Kae1 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF], the mutations on this motif paralyzed its activity in t 6 A biosynthesis of tRNA t 6 A [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. These observations suggest that the conserved metal-binding motif may play a catalytic role in transfer of threonylcarbamoyl-moeity from TCA onto A 37 of tRNA.

Bud32 is the second most conserved component of KEOPS and has orthologs in archaea and eukaryotes. Bud32 is about 25% identical in sequence to mammalian cAMP-dependent protein kinase (PKA) and possesses conserved motifs of GxGxxG and D 161 LTSSN at the ATP-binding lobe and the catalytic loop of protein kinases, respectively [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF][START_REF] Hanks | Genomic analysis of the eukaryotic protein kinase superfamily: a perspective[END_REF][START_REF] Stocchetto | Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase[END_REF]. Furthermore, sequence and structural analyses show Bud32 is more closely related to atypical RIO protein kinase superfamily whose members possess kinase activity essential for ribosome biogenesis (LaRonde-LeBlanc & Wlodawer, 2005b). Indeed, an ATP-dependent kinase activity of Bud32 has been demonstrated based on its capability to phosphorylate casein, osteopontin and Grx4 proteins as well as Kae1 [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Lopreiato | Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Stocchetto | Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase[END_REF]. In addition, S. cerevisiae Bud32 undergoes autophosphorylation at Ser 187 and Ser 189 on the catalytic loop (185)(186)(187)(188)(189)(190) and the autophosphorylation is required for activation of the Bud32 kinase activity [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF]. PRPK (p53 related protein kinase) is a human ortholog of Bud32 and phosphorylates Ser 15 of the cancer suppressor protein p53 [START_REF] Facchin | Functional homology between yeast piD261/Bud32 and human PRPK: both phosphorylate p53 and PRPK partially complements piD261/Bud32 deficiency[END_REF][START_REF] Miyoshi | Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein[END_REF].

Recently, an in vitro ATPase activity has been determined for archaea Bud32 [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. The mutation on Asp 253 of C. thermophilum Rio2, which is equivalent to the conserved Asp182 of S. cerecisiae Bud32, abolished both the kinase and ATPase activities [START_REF] Ferreira-Cerca | ATPase-dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit[END_REF]. The mutations of Asp 161 of S. cerecisiae Bud32 and Asp 127 of P. abyssi Bud32 have abolished the kinase and ATPase activities of Bud32 as well as its activity in t 6 A biosynthesis [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. These results demonstrated the catalytic importance of Asp 161 of Bud32, though it still remains unclear whether kinase activity or ATPase activity or both is required for the t 6 A biosynthesis.

Cgi121 and its human ortholog interact with Bud32 and PRPK, respectively [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Miyoshi | Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein[END_REF]. Cgi121 was identified to directly interact with Bud32 and interaction between Cgi121 and Bud32 is essential for cell life [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Ho | Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry[END_REF]. The binding of Cgi121 enhances the stability of Bud32 and may regulate its phosphotransferase activity. Indeed, it was found that Cgi121 functions to positively regulate the autophosphorylation of Bud32, during which Lys 52 , Asn 166 and Asp 182 and Asp 161 are involved [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Also in archaea, Cgi121 interacts with Bud32 and therefore potently activates the ATPase activity of Bud32 [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Likewise, human Cgi121 negatively regulates the phosphorylation of p53 by PRPK through directly interacting with PRPK in a mutually exclusive manner to p53 [START_REF] Miyoshi | Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein[END_REF]. Though deletion of CGI121 gene has less drastic effects than the deletion of the other subunits in both the biosynthesis of tRNA t 6 A and cell life, the direct interaction between Cgi121 and Bud32 is required for the in vitro biosynthesis of t 6 A in archaea [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

Pcc1 is required for cell cycle progression and mating projection formation of yeast cells and PCC1 deletion strains exhibit severe growth phenotypes (Kisseleva-Romanova et al, 2006b). Pcc1 is involved in transcriptional regulation of a set of pheromone-inducible and galactose-inducible genes. In P. furiosus, Pcc1 interacts with Kae1 and the two proteins form a tetramer in which Pcc1 functions as a dimerization module [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

Gon7 is essentially needed as one subunit of KEOPS in yeast for the biosynthesis of tRNA t 6 A and for maintaining the telomere length [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Without structural information, the sequence-based analysis shows that Gon7 has no recognizable functional homologs in fungus.

The t 6 A biosynthesis by yeast mitochondria Qri7 and Sua5

Mitochondrion have specialized ribosomes that share features with the bacterial and cytoplasmic ones [START_REF] Amunts | Structure of the yeast mitochondrial large ribosomal subunit[END_REF]. The defective mitochondrial translation results in mitochondrial dysfunction, ultimately causing growth defects of the cells [START_REF] De Luca | Mutations in yeast mt tRNAs: specific and general suppression by nuclear encoded tRNA interactors[END_REF]. Mitochondrial tRNAs contain their own set of modified nucleosides, some of which are also present in cytoplasmic tRNAs. In human mitochondria, t 6 A was found in the tRNAs for Ile, Thr, Asn and Lys [START_REF] Suzuki | A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs[END_REF]. Only a few cytosolic tRNAs such as tRNA Lys(CUU) and tRNAs Glu are found to be imported into yeast mitochondria, indicating that the biogenesis and processing of most mitochondrial tRNAs occur in mitochondria [START_REF] Schneider | Mitochondrial tRNA import and its consequences for mitochondrial translation[END_REF].

The tRNA t 6 A modification is present in both cytoplasmic and mitochondrial tRNAs. This was confirmed by analysis of the deletion of the KAE1 gene in yeast in which trace amount of t 6 A was detected and found to originate from mitochondrial tRNA [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. Consistently, the tRNA t 6 A was not detected in a strain carrying a double deletion of KAE1 and QRI7. The KAE1 deletion mutant could be partially complemented by introduction of QRI7 for the in vivo biosynthesis of t 6 A [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF]. In a recent study, mitochondrial Qri7 and Sua5 accomplished the in vitro biosynthesis of tRNA t 6 A, showing a minimal tRNA t 6 A modification system constituted by Qri7 and Sua5. Like for Kae1 in KEOPS, the mutations on the metal-binding motif of Qri7 (H145A or H149A or D361A) abolished the formation of tRNA t 6 A [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. In addition, unlike its cytoplasmic paralog Kae1 that directly interacts with both Pcc1 and Bud32, Qri7 exists instead as a homodimer, of which the disruption results in the dysfunction of Qri7 in the in vitro t 6 A biosynthesis [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Fig. 11

Proposed pathway for the minimal eukaryotic biosynthesis of tRNA t 6 A using Sua5 and Qri7. Sua5 binds threonine, bicarbonate and ATP to generate threonylcarbamoyladenylate (TCA) using its adenylation function. TCA is then released by Sua5 and bound to a Qri7 homodimer (modelled with one tRNA bound to its active site), which conjugates a threonylcarbamoyl-moiety onto bound tRNA substrates. This figure was adapted from [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Sua5 is strictly required for both the in vivo and in vitro biosynthesis of t 6 A [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The absence of the SUA5 gene in yeast mitochondrial genome suggests that the intermediate TCA is synthesized by Sua5 in the cytoplasm, followed by its diffusion into mitochondria. Recently, the yeast SUA5 ORF was shown to possess two inframe AUG codons, of which the first one initiates the translation of a mitochondrial targeting sequence [START_REF] Thiaville | Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes[END_REF]. Further analysis showed that the mitochondria-localized Sua5 is needed for cell growth on the nonfermentable carbon source glycerol and for complementation. In the in vitro t 6 A assay by Qri7 and Sua5, the mutations of residues that are putatively involved in the ATP-and threonine-binding in Sua5 (K93A, R94A, S196A, N198A, S234V and R174E) totally abrogate the formation of t 6 A [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Qri7 is capable of synthesizing tRNA t 6 A when complemented with E. coli YrdC [START_REF] Thiaville | Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes[END_REF]. The in vitro t 6 A assay further demonstrated that the biosynthesis of tRNA t 6 A proceeds in a consecutive stepwise manner: Sua5 synthesizes an unstable intermediate TCA whose threonylcarbamoylmoiety is transferred onto A 37 of tRNA by Qri7 (Fig. 11) [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Fig. 12

The proposed mechanism for the biosynthesis of tRNA t 6 A by KEOPS and Sua5 from archaea. ○ 1 Formation of TC-AMP (TCA); ○ 2 The binding of tRNA and TC-AMP to KEOPS;

○ 3 Transfer of threonylcarbamoyl-moiety to adenosine at position 37 of tRNA; ○ 4 Release of t 6 A-modified tRNA. This figure was taken from [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

The t 6 A biosynthesis by KEOPS and Sua5 in archaea

KEOPS and Sua5 from P. abyssi are necessary and sufficient for the in vitro biosynthesis of tRNA t 6 A [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Further characterization demonstrated that in the archaea t 6 A biosynthesis system strictly requires Kae1, Bud32 and Pcc1 while Cgi121 is to some extent dispensable [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. That is consistent with the observation that Cgi121 has milder effect on the in vivo formation of t 6 A in yeast cells and cell life [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Perrochia et al., characterized the molecular interactions among the components of archaea KEOPS and substrate tRNA. It was shown that the Kae1-Pcc1 sub-complex is the minimum core for tRNA binding and that Bud32 further tightens this binding. Cgi121 functions as an accessory protein that allosterically regulates the t 6 A catalytic activity of KEOPS. The mutations on the metal-binding (H107A) and ATP-binding residue (D159A) of Kae1 have abolished the formation of t 6 A [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. However, unlike yeast KEOPS that readily hydrolyzes ATP into ADP in absence of other binding partners, archaeal KEOPS hydrolyzes ATP into ADP only in presence of tRNA, suggesting that archaea KEOPS undergoes an activation process when bound to tRNA, during which the hydrolysis of ATP by Bud32 is involved (Fig. 12) [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

Like yeast Sua5, an ATP-pyrophosphatase activity of P. abyssi Sua5 was observed in presence of L-threonine. In light of the in vivo and in vitro heterologous complementation of Sua5 by its orthologs [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF], a similar two-step reaction mechanism for the biosynthesis of tRNA t 6 A in archaea has been hypothesized (Fig. 12): first, P. abyssi Sua5 utilizes ATP, L-threonine and bicarbonate to synthesize an intermediate TCA; second, the L-threonylcarbamoy-moiety is transferred from TCA onto A 37 of tRNA by KEOPS [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

The t 6 A biosynthesis by YrdC and YgjD/YeaZ/YjeE in bacteria

All the four genes, ygjD, yeaZ, yjeE and yrdC are essential for bacterial cell growth [START_REF] Abergel | Structural genomics of highly conserved microbial genes of unknown function in search of new antibacterial targets[END_REF][START_REF] Arigoni | A genome-based approach for the identification of essential bacterial genes[END_REF]Galperin & Koonin, 2004a).

The deletion of each of ygjD, yeaZ and yjeE leads to unusually elongated morphologies of E. coli cells and eventually resulted in severe growth phenotypes [START_REF] Handford | Conserved network of proteins essential for bacterial viability[END_REF][START_REF] Hashimoto | Effects on Transcription of Mutations in ygjD, yeaZ, and yjeE Genes, Which Are Involved in a Universal tRNA Modification in Escherichia coli[END_REF][START_REF] Kaczanowska | The YrdC protein--a putative ribosome maturation factor[END_REF]. In thermo-sensitive mutants of ygjD, yeaZ and yjeE, an upregulated transcription profile was observed for a set of genes whose amino acid sequence of the leader peptides contain thr-and ile-operons [START_REF] Hashimoto | Effects on Transcription of Mutations in ygjD, yeaZ, and yjeE Genes, Which Are Involved in a Universal tRNA Modification in Escherichia coli[END_REF]. Sequence-based prediction of the structure and function suggested that YgjD possesses an HSP70-actin fold and may have new type of enzymatic activity adapted from O-sialoglycoprotease activity [START_REF] Aravind | Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches[END_REF]. Indeed, a glycopeptidase activity of the essential YgjD was first demonstrated in the peptidoglycan synthesis of cell wall of S. aureus cells [START_REF] Zheng | Identification of an essential glycoprotease in Staphylococcus aureus[END_REF]. Later on, YgjD was implicated in metabolic regulation of toxic glycated proteins, modification of substrates of peptidoglycan hydrolases and regulation of hydrolases in S. aureus [START_REF] Katz | The ubiquitous conserved glycopeptidase Gcp prevents accumulation of toxic glycated proteins[END_REF][START_REF] Zheng | Conditional mutation of an essential putative glycoprotease eliminates autolysis in Staphylococcus aureus[END_REF].

YeaZ shares 29% sequence identity with YgjD within their first 100 amino acids and therefore, is classified as a paralog of YgjD [START_REF] Jeudy | Preliminary crystallographic analysis of the Escherichia coli YeaZ protein using the anomalous signal of a gadolinium derivative[END_REF].

YeaZ also possesses an HSP-70 actin fold, but lacks a metallo-protease motif (HxxxH-D), which is extremely conserved in YgjD and all its orthologs Kae1 and Qri7 [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF].

YjeE belongs to an ATPase or kinase protein family (COG0802) and is highly conserved in bacteria but has no orthologs in archaea and eukarya.

The sequence of YjeE contains a conserved ATP-binding loop of Walker A motif (GxxxxGKT/S), which is present in sequences of most ATPases, Kinases and P-loop GTPases [START_REF] Brown | Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology[END_REF][START_REF] Freiberg | Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria[END_REF]. Biochemical analysis shows that YjeE binds to both ATP and ADP and exhibits weak intrinsic ATPase activity [START_REF] Allali-Hassani | Probing the active site of YjeE: a vital Escherichia coli protein of unknown function[END_REF]. The B. subtilis YdiB is an ortholog of E. coli YjeE and also possesses very weak ATPase activity, which is related to its in vivo function [START_REF] Karst | The ATPase activity of an 'essential' Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization[END_REF]. However, based on its structural similarity to a number of GTPases, YjeE has been speculated to be a molecular switch, which is often involved in a multiprotein signaling pathway [START_REF] Brown | Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology[END_REF]. Whether the nucleotide-binding property or the ATPase activity of YjeE is accountable for its essential role in cell life is still unknown. YeaZ engages in partner switching in response to an undefined signal, binding with YjeE instead, and free YgjD can then accumulate within the cell. ATP binding or hydrolysis by YjeE is thought to reverse the partner switch, allowing YeaZ to form a complex with YgjD, which is then subjected to proteolysis. The figure C was taken from [START_REF] Msadek | Grasping at shadows: revealing the elusive nature of essential genes[END_REF].

In E. coli, the biosynthesis of tRNA t 6 A also requires YeaZ and YjeE [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF], which are not orthologs of any KEOPS subunits. However, it was identified that YeaZ and YjeE carry out the functions of Bud32, Cgi121 and Pcc1 in the in vitro biosynthesis of tRNA t 6 A [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF]. Likewise, YdiE, YdiC, YdiB and YwlC, which are the B.

subtilis orthologs of YgjD, YeaZ, YjeE and YrdC, respectively, are necessary and sufficient for the in vitro biosynthesis of tRNA t 6 A [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF]. Moreover, YrdC could substitute YwlC in the t 6 A assay by proteins from B. subtilis.

From studies in E. coli and B. subtilis, a common scheme for the t 6 A biosyn-thesis can be proposed in bacteria: YwlC (YrdC) catalyzes the TCA biosynthesis and YgjD, YeaZ and YjeE functions cooperatively in transfer of the threonylcarbamoyl-moiety from TCA to the substrate tRNA [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. As desirable, YrdC has been shown to bind ATP and L-threonine, the two starting substrates for biosynthesis of t 6 A [START_REF] Harris | YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase[END_REF]. In addition, an ATP-pyrophosphatase activity of YrdC was observed in presence of L-threonine and bicarbonate [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF]. Chemically, the mixture of YgjD, YeaZ and YjeE was shown to hydrolyze ATP into ADP, which is, like KEOPS in yeast and archaea, uncoupled to the TCA biosynthesis reaction. However, the preferential binding of an unmodified anticodon stem loop of cognate tRNA to YrdC was also determined [START_REF] Harris | YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase[END_REF][START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. Of particular note, the analysis of the specific binding by saturation transfer difference (STD) NMR showed the interaction between YrdC and the tRNA ASL involves four nucleotides on the anticodon stem loop [START_REF] Harris | YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase[END_REF]. This observation is puzzling since it was convincingly shown that in B. subtilis YwlC could be physically uncoupled to YdiE, YdiC and YdiB in biosynthesis of t 6 A. Or alternatively, YrdC is capable of interacting with tRNA when it's bound to YgjD/YeaZ/YjeE individually or together.

Unlike KEOPS protein complex, there have not been any reports on the formation of a stable complex of YgjD, YeaZ and YjeE. The genetic and functional interaction have been identified and predicted for ygjD, yeaZ and yjeE genes. For example, genes ydiE, ydiC, ydiB are clustered and share one operon in genome of B. subtilis and ydiC is localized between ydiB and ydiD (Fig. 13A) [START_REF] Dandekar | Conservation of gene order: a fingerprint of proteins that physically interact[END_REF][START_REF] Oberto | SyntTax: a web server linking synteny to prokaryotic taxonomy[END_REF][START_REF] Wolf | Genome alignment, evolution of prokaryotic genome organization, and prediction of gene function using genomic context[END_REF]. In addition, B. subtilis ydiB was identified to interact with a number of genes in both genomic context and functional pathways (Fig. 13B). The genetic complementation of E. coli ygjD could not be accomplished by B. subtilis YgjD, S. cerevisiae KAE1 and QRI7, but B. subtilis ygjD-yeaZ, demonstrating that a very specific molecular recognition is needed for the association of YgjD and YeaZ [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF]. Characterization of the bacterial protein-protein interaction network showed that YgjD interacts with YeaZ, forming a heterodimer that might be involved in ribosome function, RNA processing, and/or RNA binding [START_REF] Butland | Interaction network containing conserved and essential protein complexes in Escherichia coli[END_REF]. In line with the possible existence of an interaction network of YgjD-YeaZ-YjeE, indeed, the bacterial two-hybrid experiments and in vitro protein-protein interaction analysis showed that YeaZ interacts with either YgjD or YjeE in a mutually exclusive manner, forming an interaction network of cell signaling (Fig. 13C) [START_REF] Handford | Conserved network of proteins essential for bacterial viability[END_REF][START_REF] Msadek | Grasping at shadows: revealing the elusive nature of essential genes[END_REF].

However, the attempt to purify the putatively preassembled binary YjeE-YeaZ proved unsuccessful, demonstrating that the interaction is too weak to persist throughout purification or other binding cofactors are required. Recently, an interaction between YjeE and the heterodimer YgjD-YeaZ from S. typhimurium in presence of ATP/ADP was observed [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF], though no information on the quaternary structure of the associated proteins.

The structure-function relationship of the tRNA t 6 A modifying enzymes

High-resolution protein structures provide unparalleled molecular details for investigating the underlying mechanisms of proteins. The determination of the crystal structures of enzymes that are involved in the tRNA t 6 A modification has advanced the molecular elucidation of their roles in biosynthesis of t 6 A as well as their functions implicated in other biological aspects.

The structural characterization of the YrdC/Sua5 family proteins

The crystal structure of E. coli YrdC was first determined and it shows that YrdC adopts an α/β twisted mixed parallel and antiparallel open-sheet structure [START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. The topology of YrdC reveals a very unusual order of the seven α-helices and seven β-strands consisting of three parts: the first is composed of α1, β1, β2 and α2; the second comprises β3, α3, α4, β3, α5

and β5; and the third contains α6 and β6. The C-terminal α7 and β7 folds over the remainder of the domain (Fig. 14A and14B). The two conserved motifs, K 50 xR 52 and S 139 xN 141 , form a KRSN tetrad in the putative active site (Fig. 14A). The most striking structural feature is a large concave surface formed by β3, β4, β5, β7, and α5. The electrostatic surface potential of this concave depression is positive, consistent with its binding properties to doublestranded RNA, tRNA as well as various tRNA anticodon stem loops [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Harris | YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase[END_REF][START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. Meanwhile, E.

coli YciO was classified as one member of the Sua5/YrdC protein family for its sequence conservation and structural similarity, shown by a sequence identity of 27% and an RMSD of 1.52 Å over 131 C α atoms aligned to YrdC [START_REF] Jia | Crystal structure of the YciO protein from Escherichia coli[END_REF][START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. YciO also has an α/β architecture that is composed of 6 α-helices and 10 β-strands and the bimodal distribution of YciO is less pronounced than that of YrdC (Fig. 14A and14C). However, the molecular surface and sequence composition in the putative active site are quite different, as typically represented by the absence of the KRSN tetrad, which instead are RLSM at the equivalent positions in YciO (Fig. 14A). The structural features of YciO including the α/β folds and the surface charge distribution favor its binding to RNA molecules. However, t 6 A biosynthesis in S. cerevisiae SUA5 deletion cells could be restored introduction of either E. coli yrdC gene or B. subtilis ywlC gene, but not E. coli yciO gene, suggesting YciO is not involved tRNA t 6 A modification [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF]. The structure of S.

tokodaii Sua5 shows that Sua5 is composed of an N-terminal YrdC and a Cterminal Sua5 domain (Fig. 14D). The structure of the N-terminal YrdC domain is very similar to that of YrdC from E. coli, shown by an RMSD of 1.6Å over 184 C α atoms. Sua5 also has the three conserved motifs: KRSN tetrad motif [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF][START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF]. The C-terminal Sua5 domain consists of 3 α-helices and 5 β-strands and adopts a Rossmann fold. Interestingly, Sua5 domain is structurally close to M. jannaschii HypB and T. aquaticus Fth, both of which are GTP-binding proteins [START_REF] Freymann | Functional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP[END_REF][START_REF] Gasper | Structural insights into HypB, a GTP-binding protein that regulates metal binding[END_REF]. In two crystal structures of S. tokodaii Sua5, however, the 20-residue loop linking the YrdC and Sua5 domains was not observed due to its flexibility in the crystals (Fig. 14D).

Sua5/YrdC utilizes ATP, L-threonine and bicarbonate to synthesize an unstable intermediate threonylcarbamoyladenylate (TCA). The binding of ATP, L-threonine has been quantified for S. tokodaii Sua5 and E. coli YrdC [START_REF] Harris | YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase[END_REF][START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF]. Structures of S. tokodaii Sua5 in complex with L-threonine, AMPPNP or TCA were determined (Fig. 14E) [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF][START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF][START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF] [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF][START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF][START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF].

The mutations on the KRSN tetrad (Lys 93 , Arg 95 , Ser 196 and Asn198) of S.

cerevisiae Sua5 have abolished its activity in TCA biosynthesis [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

2.3.2

The structural characterization of Kae1/Qri7/YgjD family proteins P. abyssi Kae1 consists of two domains (domain I and domain II as shown in Fig. 15A), each possessing a βββαβαβα topology. One AMPPNP and two metal ions, Fe 3+ and Mg 2+ , are bound at the active site situated at the interface between the two domains (Hecker et al, 2007b). S. typhimurium YgjD is structurally similar to Kae1 with an RMSD of 1.9 Å over 321 Cα atoms (Fig. 15) [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. Also S. cerevisiae Qri7 is very similar to Kae1 and YgjD with RMSDs of 2.0 Å and 2.2 Å over 327 Cα atoms of P. abyssi Kae1 and 337 Cα atoms of S. typhimurium YgjD, respectively (Fig. 15C) [START_REF] Tominaga | Structure of Saccharomyces cerevisiae mitochondrial Qri7 in complex with AMP[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Compared to the prototypical ASKHA family proteins, Kae1/Qri7/YgjD has two unique inserts that comprise a long intervening linker between helices α4, α5 and α6 as well as C-terminal tail adopting an extended conformation interspersed by two short 3 10 helices.

The structures of Kae1 and its orthologs in complex with nucleotides have identified the nucleotide-binding site of the Kae1/Qri7/YgjD family members.

The nucleotide is sandwiched between the domains of Kae1/Qri7/YgjD and involves two conserved loops flanked on either side by long β-strands (Fig. 15B). In P. abyssi Kae1, the adenosine-moiety of AMPPNP interacts with Asn 257 , Gly 172 and Asp 159 while the tri-phosphates are coordinated by main chain amide from Asp 285 , Gly 130 and a number of other groups of His 107 , Tyr 127 and Ser 129 (Hecker et al, 2007b). In the S. typhimurium YgjD in complex with ATPγS, the adenosine-moiety is coordinated by Asn 272 , Asp 167 and Gly 163 , the triphosphates are coordinated by Gly 268 , Ser 163 , His 115 , Asp 300 and His 139 [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. The Qri7-AMP complex structure reveals that the AMP is coordinated by the conserved residues of Asn 329 , Asp 203 , Gly 325 , D 361 , H 145 and Gly 173 [START_REF] Tominaga | Structure of Saccharomyces cerevisiae mitochondrial Qri7 in complex with AMP[END_REF]. In summary, the ATP-binding modes and sites are very conserved in the Kae1/Qri7/YgjD proteins (Fig. 15B and15D). Mutations of the ATP-binding site of S. cerevisiae Kae1 (Gly 190 , Lys 27 , Asp 213 and Asn 315 ) have abolished the ATP-binding, leading to severe growth phenotypes [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The mutations of the AMP-binding residues Arg 207 and Ser 258 of S. cerevisiae Qri7 abolish the formation of tRNA t 6 A [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. and Zn 2+ ions are represented as green and orange spheres, respectively) and the ribbon is colored according to sequence conservation within the Kae1/Qri7/YgjD family proteins (the conservation scale is given in the key at the bottom). C. The structural superposition of Kae1 (colored in red), YgjD (colored in wheat) and Qri7 (colored in slate) and the two inserts compared to ASKHA family proteins are colored in green and yellow, respectively. D. The conserved Fe-binding motif and ATP-binding residues from P. abyssi Kae1 (residue numbering), YgjD and Qri7.

Strikingly, the active sites of the Kae1/Qri7/YgjD proteins are characterized by the binding of divalent metal ions at two positions in vicinity of ATP. A conserved aspartic acid and two or three water molecules coordinate the Mg 2+ ion, which is ~3 Å away to the α-phosphate of AMP or ATP (Hecker et al, 2007b;[START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF][START_REF] Tominaga | Structure of Saccharomyces cerevisiae mitochondrial Qri7 in complex with AMP[END_REF]. The second metal ion is tightly coordinated by a totally conserved HxxxH-D motif. This type of motifs is frequently implicated in binding of Fe, Zn as well as Co [START_REF] Harding | Mespeus--a database of metal interactions with proteins[END_REF][START_REF] Harding | Metals in protein structures: a review of their principal features[END_REF]. The metal ion was Fe 3+ in P. ab-yssi Kae1 but Zn 2+ in both S. typhimurium YgjD and S. cerevisiae Qri7 (Hecker et al, 2007b;[START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF][START_REF] Tominaga | Structure of Saccharomyces cerevisiae mitochondrial Qri7 in complex with AMP[END_REF]. The nucleotides are part of the coordination sphere of both metals. It should be mentioned that only in the case of P. abyssi Kae1 the nature of the bound metal Fe 3+ was further experimentally determined by electron paramagnetic resonance, for the YgjD and Qri7 crystal structures, the assignment of bound metals was based on electron density criteria. The reason for this discrepancy in the nature of the bound metal ions and the pertinence for the activity of the Kae1/Qri7/YgjD orthologs have not been properly addressed and remains an interesting question.

Mutations of the metal-binding motif of S. cerevisiae Kae1 (His 141 and His 145 ), P. abyssi Kae1 (His 107 ) and S. cerevisiae Qri7 (His 145 , His 149 and Asp 361 ) have totally abolished t 6 A activity (Hecker et al, 2007b;[START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Moreover, the mutations of Asp 43 and Asp 44 , which may be involved in coordination of Mg 2+ , as based on its structural conservation to S. typhimurium YgjD, also abrogated the t 6 A activity of Qri7 [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. It is for the moment not known whether the metal ions may play a direct role in the catalytic activity and/or whether they are involved in stabilizing the bound conformation of substrates.

Fusion of Kae1 domain and YrdC domain in TobZ and HypF

Considerable mechanistic insight of the Kae1/YgjD/Qri7 family came from comparison with enzyme families that catalyze related carbamoylation reaction but using totally different substrates. S. tenebrarius TobZ is involved in the maturation of 2-deoxystreptamine-containing aminoglycoside antibiotics, while E. coli HypF participates in the synthesis of active [NiFe] hydrogenases [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF][START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF]. Both enzymes are fusion proteins of YrdC and Kae1 orthologs (Fig. 16). The YrdC and Kae1 subunits catalyze two consecutive reactions: formation of a carbamoyladenylate and transfer of the carbamoyl-group to acceptors. The O-carbamoyltransferase TobZ belongs to the BcrAD_BadFG superfamily and carbamoylates the antibiotic tobramycin to form nebramycin [START_REF] Kharel | Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius[END_REF]. TobZ-YrdC utilizes carbamoylphosphate and ATP to form carbamoyladenylate, of which the car-bamoyl-moiety is subsequently transferred to the 6'-hydroxyl group of the acceptor tobramycin by TobZ-Kae1 (Fig. 16A). The whole reaction produces one molecule of phosphate and one molecule of pyrophosphate [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF]. HypF-YrdC and HypF-Kae1 domain in the S-carbamoyltransferase are in a reverse order compared to that in TobZ. Nevertheless, the YrdC domain utilizes carbamoylphosphate and ATP to catalyze the formation of carbamoyladenylate, of which the carbamoyl-moiety is subsequently transferred to the C-terminal cysteine of HypE by Kae1-like domain (Fig. 16B) [START_REF] Petkun | Structure of hydrogenase maturation protein HypF with reaction intermediates shows two active sites[END_REF]. finger-like domain, a HypF-YrdC and a C-terminal HypF-Kae1 unit (Fig. 17E and 17F) [START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF]. In particular, both the metal binding mo- tif HxxxH-D from Kae1/YgjD/Qri7 and the KRSN tetrad motif from Sua5/YrdC family are also conserved in Kae1-like domain and YrdC-like domain of TobZ and HypF, respectively (Fig. 17A-D). Moreover, the binding modes of AMPPNP in P. abyssi Sua5 and AMPCPP in TobZ-YrdC are very similar. The same holds for the binding mode of AMPPNP/ATPγS/AMP in Kae1/YgjD/Qri7 and carbamoyladenylate in TobZ-and HypF-Kae1 subunits [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF][START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF]. The catalytic centers accommodated by the YrdC and Kae1 subunits are 16-20 Å apart in both TobZ and HypF, and are connected by the above-mentioned cavity. The presence of this tunnel probably provides a catalytic advantage: the chemically labile carbamoyladenylate can be directly channeled to the acceptor substrate preventing premature disintegration. Interestingly, Kae1 and full length Sua5 were found to be fused in YP_008438716.1 and encoded by gene ps_2314 from Pandoravirus salinus (Fig. 17E) [START_REF] Philippe | Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes[END_REF]. It remains an intriguing question why this option was not retained for the t 6 A enzymatic system. Although the TCA intermediate is labile, YrdC and Kae1 are not covalently linked. It cannot be excluded that YrdC/Sua5 and YgjD/Kae1 form a transitory complex during the t 6 A synthesis but there is presently no experimental evidence for such a complex.

The structural model of KEOPS

The crystal structure of M. jannaschii fusion protein MJ1130 that is composed of N-terminal Kae1 and C-terminal Bud32 revealed M. jannaschii Kae1 is structurally identical to P. abyssi Kae1 (Hecker et al, 2007b;[START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF]. The M. jannaschii Bud32 consists of an N-lobe with a five-stranded βsheet flanked by α-helix and a C-lobe with two β-strands and five α-helices.

The Bud32 represents a kind of 'minimal kinase' structure and the C-lobe lacks the infrastructure responsible for phosphoacceptor site recognition [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The crystal structure of the ternary complex Kae1-Bud32-Cgi121 from M. jannaschii revealed that Cgi121 is composed of a central antiparallel β-sheet abutted by two and five α-helices on either face. In this ternary complex model, Cgi121 binds to the C-lobe of Bud32 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The crystal structure of T. acidophilum Kae1 in complex with P. furiosus Pcc1 reveals Pcc1 is a KH-like domain, a large protein family that is functionally implicated in binding of RNA, ssDNA and hairpin RNA (Fig. 18) [START_REF] Valverde | Structure and function of KH domains[END_REF].

The ensemble of the KEOPS subcomplexes permits reconstituting the complete archaea KEOPS protein complex, in which Pcc1, Kae1, Bud32 and Cgi121 are linearly arranged (Fig. 19) [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The C-terminal domain of Kae1 protrudes into the cleft of two lobs of Bud32, forming a compact binary complex. While Cgi121 contacts Bud32 on its N-lobe, Pcc1 contacts Kae1 at its N-terminal domain. Combining biochemical and characterization and structural data, it has been demonstrated that archaea KEOPS exists as a dimer of tetramers in which Pcc1 functions as a dimerization module (Fig. 18 and Fig. 19) [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

Based on the structural model of archaea KEOPS, mutations that were designed to disrupt the interactions between Pcc1 and Kae1, Kae1 and Bud32, Bud32 and Cgi121 in S. cerevisiae have led to lethal phenotypes, suggesting that Pcc1, Kae1, Bud32 and Cgi121 of yeast KEOPS also assemble in manner that is similar to those of archaea KEOPS [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Furthermore, it was suggested that the yeast KEOPS/EKC might exist as dimer or trimer of the hetero-pentameric complex because the molecular weight from gel filtration experiments was estimated to be 300 kD and 400 kD, respectively [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]Kisseleva-Romanova et al, 2006b). However, the structure of Gon7 and the quaternary structure of KEOPS are presently not known.

As delineated in the archaea KEOPS model (Fig. 19), the absence of direct interaction between Cgi121 and Kae1 may explain the milder effect of the deletion of CGI121 gene in t 6 A biosynthesis [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Thus, the contributing role of Cgi121 in t 6 A biosynthesis could be ascribed to its positive role of activating the autophosphorylation of Bud32 and therefore, its negative regulation of the ATPase activity of Bud32 [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Miyoshi | Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. In contrast, Pcc1 is strictly required to form a complex with Kae1, providing a scaffold for binding the tRNA. The binding of Bud32 to the Kae1-Pcc1 complex also contributes to the increase binding affinity of tRNA [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Combining the biochemical data, the archaea KEOPS model shows that the ternary complex Pcc-Kae1-Bud32 is the core, while Cgi121 functions as an allosteric regulator of the catalytic activity of Kae1 in t 6 A biosynthesis.

Fig. 19 The schematic representation of the molecular architecture of the dimer of heteropentamer KEOPS from archaea. The figure was adapted from [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

The essential role of protein assembly in t 6 A biosynthesis

Though the YrdC and Kae1 subunits are fused in the TobZ/HypF enzymes to catalyze the carbamoyladenylation reaction, such a direct interaction is also anticipated for Sua5/YrdC and KEOPS/Qri7/YgjD to expedite the whole reaction t 6 A biosynthesis. However, it has been clearly demonstrated that a direct interaction between S. cerevisiae Sua5 and Qri7 is not strictly required in the in vitro assay, neither is that for B. subtilis orthologs of YrdC and YgjD. Nonetheless, the intermolecular interactions of the KEOPS subunits play a crucial role in regulation of its activity supporting the cell life. While mutations that disrupt the interaction between Kae1 and Pcc1 or Bud32 resulted in severe growth phenotypes comparable to deletion of each subunit, mutation disrupting the interaction between Bud32 and Cgi121 caused less drastic growth phenotype [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The weaker effect of Cgi121 deletion for cell life is in good agreement with the non-essentiality of Cgi121 for the t 6 A synthesis in yeast and archaea [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. The phenotypes and biochemical effects caused by the deletion of or mutations on each component of the yeast KEOPS were explored and summarized in Table 2.

The essentiality of protein-protein interaction for t 6 A biosynthesis is best represented by the homodimer Qri7. The crystal structure of Qri7 reveals that the dimerization interface involves a large hydrophobic surface contributed by residues on the α1-and α2-helices from the N-terminal domains. Mutations of Val 134 and Lys 130 that have disrupted the homodimerization of Qri7 abolish not only the t 6 A activity but also its capability to rescue the growth of the KAE1 deletion strain [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The structural superposition of the Qri7 homodimer with Kae1 of archaean KEOPS revealed large steric clashes between Bud32 and the C-terminal domain of Qri7, implicating Bud32 in regulating Kae1 by direct interaction, or vice versa [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. This comparison further reveals that the interfaces used by the homodimer Qri7 and the binary complex Kae1-Pcc1 are identical and that they use similar secondary structure elements (Fig. 20) [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The homodimerization of Qri7 is evolutionarily adapted for mitochondrial to overcome the missing partner to create a scaffold for binding of tRNA with regard to its role in t 6 A biosynthesis.

The crystal structures of YeaZ have revealed that YeaZ also adopts a twodomain architecture characteristic of HSP70/actin-like fold. YeaZ exists as a homodimer in solution and the dimeric interface is formed by α1 and α2 helices from both subunits, which form a 4-helical bundle [START_REF] Aydin | Structural analysis of the essential resuscitation promoting factor YeaZ suggests a mechanism of nucleotide regulation through dimer reorganization[END_REF][START_REF] Nichols | Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog[END_REF][START_REF] Stelter | A complement to the modern crystallographer's toolbox: caged gadolinium complexes with versatile binding modes[END_REF][START_REF] Xu | Structure of an essential bacterial protein YeaZ (TM0874) from Thermotoga maritima at 2.5 A resolution[END_REF]. Analysis of sequence conservation of YeaZ from bacteria reveals an absolutely conserved glycinerich motif 65 GPGXXTGXR, which is located at the cleft between the two domains. It was proposed but not observed that this could be a nucleotidebinding site [START_REF] Aydin | Structural analysis of the essential resuscitation promoting factor YeaZ suggests a mechanism of nucleotide regulation through dimer reorganization[END_REF]. The crystal structure of the heterodimer YgjD-YeaZ from S. typhimurium reveals that the N-terminal α2 and α3 helices of YgjD form an interacting 4-helical bundle with N-terminal α1 and α2 helices of YeaZ, preventing YeaZ from forming a homodimer. In addition, 11 C-terminal residues that were disordered in the YeaZ homodimer are well structured and involved in an interaction with YgjD [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. The N-terminal α1 helix of YeaZ interacts with the C-terminal tail of YgjD, which was previously shown to be essential for its interaction with YeaZ in S. aureus [START_REF] Lei | The Cterminal domain of the novel essential protein Gcp is critical for interaction with another essential protein YeaZ of Staphylococcus aureus[END_REF]. In comparison to the YeaZ from the homodimer, YeaZ from the hetero-

Is the ancient KEOPS machinery involved in other cellular processes?

Recent studies further showed that KEOPS is also conserved and present in fly and human, though with compositional variations (Fig. 21). Drosophila CGI121 and LAGE3, the human ortholog of PCC1 [START_REF] Costessi | The Human EKC/KEOPS Complex Is Recruited to Cullin2 Ubiquitin Ligases by the Human Tumour Antigen PRAME[END_REF]. The human KEOPS interacts directly with the human oncoprotein PRAME, a component of Cullin2-based ubiquitin ligases. In yeast, essential roles of KEOPS in telomere elongation and transcriptional regulation have been reported in the pioneering work [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]Kisseleva-Romanova et al, 2006b). Interestingly, the programmed -1 ribosomal frameshifting has been found to control the cellular abundance of the yeast telomerases Est1 and Est2 as well as telomerase recruiting proteins Stn1 and Cdc13 [START_REF] Advani | Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsensemediated mRNA decay pathway[END_REF], raising a possibility to ascribe the function of KEOPS in telomere elongation to its primordial role in tRNA t 6 A modification. However, it still remains to be ascertained that the short telomere and unregulated transcription in KEOPSdeletion and mutant strains could be accounted by the defective tRNA t 6 A modification. Or alternatively, KEOPS does participate in a rather wide range of biological pathways that are correlated with telomere replication apparatus as well as transcription machinery.

Fig. 21 The distribution and composition of KEOPS complex in archaea and eukarya.

KEOPS in telomere decapping and elongation

Telomeres shorten with cycles of cell division due to the genome replication. The incomplete length of telomere leads to a range of catastrophic effects on the fundamental pathways of cells [START_REF] Smogorzewska | Regulation of telomerase by telomeric proteins[END_REF]). The telomere length maintenance and homeostasis is a complex dynamic process in which dozens of major proteins are involved [START_REF] Malyavko | Telomere length regulation in budding yeasts[END_REF][START_REF] Smogorzewska | Regulation of telomerase by telomeric proteins[END_REF]. To ensure the viability, cells need not only to protect the telomeres from chemical damage, but also to maintain the integrity of the telomeres via replication of the single stranded DNA ends by a dedicated DNA-polymerase called telomerase, which contains a highly conserved telomerase reverse transcriptase (TERT) and an associated template telomerase RNA (TR) domain. The short GC-rich repeats of most eukaryotic telomeric DNA has a 3' single-stranded DNA overhang, which is a specific binding sequence for several telomere capping proteins such as Cdc13, Stn1 and Ten1 [START_REF] Martin | Protection of telomeres by a conserved Stn1-Ten1 complex[END_REF][START_REF] Smogorzewska | Regulation of telomerase by telomeric proteins[END_REF]). In S.

cerevisiae, the ternary complex Mre11/Rad20/Nbs1, Sae2 and Sgs1 participate in the multiple pathways involved in generation of the 3' overhang of telomere [START_REF] Bonetti | Multiple pathways regulate 3' overhang generation at S. cerevisiae telomeres[END_REF][START_REF] Nicolette | Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA doublestrand breaks[END_REF] during which the recruitment of telomerase is necessarily mediated by the Cdc13-Ten1-Stn1 proteins [START_REF] Martin | Protection of telomeres by a conserved Stn1-Ten1 complex[END_REF][START_REF] Pennock | Cdc13 delivers separate complexes to the telomere for end protection and replication[END_REF]. Cdc13 plays an essential role in mediating telomere replication by recruiting telomerase as the result of an interaction between the high affinity DNA binding domain of Cdc13 and telomeric DNA [START_REF] Pennock | Cdc13 delivers separate complexes to the telomere for end protection and replication[END_REF]. Cdc13 binds to telomeric DNA at single stranded TG 1-3 repeats [START_REF] Lin | The Saccharomyces CDC13 protein is a singlestrand TG1-3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo[END_REF] and interacts with Stm1, which in turn binds to telomeric G-quadruplex [START_REF] Hayashi | STM1, a gene which encodes a guanine quadruplex binding protein, interacts with CDC13 in Saccharomyces cerevisiae[END_REF]. The deletion of CDC13 from genome results in both shortened or aberrant elongated telomeres, apart from the RAD9-dependent cell cycle arrest at G2 stage [START_REF] Chandra | Cdc13 both positively and negatively regulates telomere replication[END_REF]. While cdc13-3 and cdc13-5, two alleles of CDC13, are implicated in defective elongation of telomere [START_REF] Chandra | Cdc13 both positively and negatively regulates telomere replication[END_REF], the cdc13-1 and cdc13-2 alleles are causing pronounced shortening of the telomere sequences [START_REF] Nugent | Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance[END_REF]. The cdc13-1 is frequently used for screening of genes that are capable to counteracting its function in capping the telomere. In a genome-wide screening for genes involved in suppressing the telomere-capping in S. cerevisiae, CGI121 (YML036W) was identified as a novel gene involved in telomere healing, together with four other functionally-recognizable genes-PIF1, EBS1, BMH1 and SAN1 [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]. The study that led to the finding that the deletion of CGI121 suppresses the cdc13-1 mutation, further found that the deletions of KAE1, BUD32, GON7

and PCC1 suppress the telomere capping defects more strongly than deletion of CGI121 [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Based on the structural model of archaea KEOPS complex, the effects of mutations of the subunits on telomere elongation have been explored (Table 2). Mutations that disrupt the interaction between Kae1 and Bud32 have resulted in short telomeres as observed for the KAE1 or BUD32 deletion strains. The mutation of Asp161 that is a determinant of kinase and ATPase activities of Bud32 has resulted in short telomeres to the same extent as for the deletion of BUD32

gene. These data suggest that the enzymatically active KEOPS complex is required to counteract the telomere shortening in the cdc13-1 strain [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The mechanistic characterization of the role of KEOPS in telomere maintenance suggested two possible mechanisms of action: first, KEOPS promotes resection of uncapped telomeres in cdc13-1 cells and therefore, facilitates the Est1 activating process in S phase; the other, KEOPS promotes the formation of 3' TG overhang at telomere in S phase, which would subsequently lead to dysfunction of the resection in cdc13-1 cells (Fig. 22) [START_REF] Bianchi | The KEOPS complex: a rosetta stone for telomere regulation?[END_REF]. S. cerevisiae Sua5 was also reportedly implicated in telomere length maintenance [START_REF] Meng | Sua5p a single-stranded telomeric DNA-binding protein facilitates telomere replication[END_REF]. Surprisingly, the mutations of the Lys 93 and Arg 95 of Sua5, which inactivate Sua5 in biosynthesis of tRNA t 6 A, also lead to the short telomeres phenotype to the same extent as the deletion of SUA5 [START_REF] Meng | Sua5p is required for telomere recombination in Saccharomyces cerevisiae[END_REF]. For the moment, no convincing data are available proving that KEOPS is directly involved in the telomere length regulation. Given the mutations of Sua5 that abolish TCA biosynthesis also lead to short telomeres in S. cerevisiae, it could be that these are indirect effects created by the absence of the t 6 A modification in the concerned KEOPS mutants, which may provoke aberrant translation and a plethora of cellular dysfunctions.

KEOPS in transcriptional regulation of essential genes

Gene transcription is a result of combined interactions between chromatin, transcription factors and a vast number of cofactors. In eukaryotes, the gene transcription is carried out in a spatiotemporal fashion by polymerases, which transiently assemble with many additional cofactors to recognize different promoters of genes [START_REF] Struhl | Fundamentally different logic of gene regulation in eukaryotes and prokaryotes[END_REF]. Transcription factors enable the polymerases to recognize different promoters, to receive different regulatory signals and to transcribe different classes of genes. Transcription is initiated when one transcription factor binds the promoter of a gene, followed by a series of cascade-like interactions among multiple proteins (activators, regulators, and repressors) at the same site. Ultimately, a transcription complex is formed at the promoter that facilitates binding and transcription by RNA polymerase [START_REF] Lee | Transcription of eukaryotic protein-coding genes[END_REF]. Chromatin structure contributes to the varying levels of complexity in gene regulation. It allows simultaneous regulation of functionally or structurally related genes that tend to be present in widely spaced clusters or domains on eukaryotic DNA [START_REF] Sproul | The role of chromatin structure in regulating the expression of clustered genes[END_REF]. A strong evidence of the implication of KEOPS in transcription is the manifestation of an association of Pcc1 with the chromatin of RNA polymerase II transcribed genes (Kisseleva-Romanova et al, 2006b).

The deletion and mutation of the PCC1 gene lead to defective expression of several genes regulated by the α-factor and galactose. The PCC1 thermosensitive mutant cells are unable to respond to pheromone probably due to transcriptional defects of a set of essential genes including FUS3, STE2, FAR1 and GAL1 (Kisseleva-Romanova et al, 2006b). Chromatin Immunoprecipitation (ChIP) analysis showed that Pcc1 associates strongly with FUS3, STE2, FAR1 and FUS1 only upon transcriptional induction, but poorly with the intergenic non-transcribed regions. In addition, Pcc1 was shown to bind to the heat-inducible HSP104 gene region in a transcription-dependent manner. By contrast, Pcc1 is persistently bound to the region of PMA1 gene, which is constitutively expressed in cells. Further analysis indicated that Pcc1 is required for efficient recruitment of SAGA and Mediator co-activators to the promoter of the GAL1 gene. The genetic interaction between KEOPS and genes such as RBP1 and RBP4 encoding for components of transcription apparatus has been observed. In that study it was also demonstrated that Pcc1, Gon7, Kae1 and Bud32 are recruited to several genomic loci upon transcription induction, suggesting that KEOPS protein complex is involved in transcriptional regulation through efficient recruitment of transcriptional coactivators. In KEOPS thermo-sensitive mutants, the transcriptome profile was monitored and analyzed, demonstrating that ~600 genes were up regulated and ~300 genes were down regulated (Daugeron et al, 2011b). The transcription profiles showed that a number of genes that are involved in biosynthesis of amino acids were up regulated. KEOPS is implicated in transcription regulation, which eventually exerts effect on the translation of the genes, such as Thr1, Thr4 and GCN4. In line with this functional implication, the drosophila KEOPS was also reported to play a role in protein homeostasis via interacting with the TOR signaling pathway [START_REF] Rojas-Benitez | The Drosophila EKC/KEOPS complex: Roles in protein synthesis homeostasis and animal growth[END_REF].

Aims and Objectives

Despite the wealth of structural data on the subunits of the KEOPS complex, we are still missing crucial information for a full understanding of the biosynthesis of tRNA t 6 A. We expect that more structural data would also shed light on the other potential biological functions of the complex such as transcriptional and telomere length regulation. For the moment there is no structure of the complete KEOPS complex and there remain uncertainties about its quaternary organization, which may be species-dependent. Fungi have a fifth KEOPS component, Gon7, which is essential for cell viability and t 6 A biosynthesis. Very few is known about this protein, which is predicted to be largely unstructured. The precise contribution of each subunits of the KEOPS complex to the overall reaction mechanism remains largely unknown. For instance, the presence of the small atypical protein kinase Bud32 is very intriguing, knowing that the transfer of the threonylcarbamoyl-moiety onto A 37 of tRNA does not chemically require the phosphorylation of a protein partner nor the hydrolysis of ATP. The biochemical role of all the subunits in the KEOPS complex would certainly became clearer if we would obtain a structure of the tRNA-KEOPS complex. The crystal structure of such a complex is beyond doubt the most important objective for the mechanistic understanding of KEOPS.

The current status about the knowledge of the bacterial t 6 A biosynthesis system is comparable to that in yeast and archaea. The characterization of the previously identified interaction network of YgjD, YeaZ and YjeE is critical to elucidate their respective roles in biosynthesis of tRNA t 6 A. The structures of the individual YgjD, YeaZ and YjeE subunits are known but not those of their mutual complexes. As for ATPase activity of Bud32, the need of the ATPase activity carried by YjeE for t 6 A activity is not understood.

The comprehension of the TCA biosynthesis is at a more advanced stage.

The structures of Sua5/YrdC proteins in complex with substrates of t 6 A biosynthesis were very informative and provided valuable insights about the biosynthesis of TCA. Why there exist of two types of TCA synthases, a 'short' form (YrdC) and a long form (YrdC/Sua5) is not clear since YrdC alone is suf-ficient for the TCA synthesis in vitro and in vivo. For my thesis work we particularly focused on the structure of the linker between N-terminal YrdC domain and C-terminal Sua5 domain, suspecting it could provide information on the collaboration between the two domains during the reaction cycle.

Operational Outlines

In order to answer the above stated questions, we conceived the following experimental work-plan.

1) Biophysical characterization of yeast KEOPS

--To determine the quarternary structure of KEOPS and subunits in solution;

--To determine the X-ray crystal structure of KEOPS;

--To determine the X-ray crystal structures of Bud32 in complex with nucleotides;

--To determine the X-ray crystal structures of Gon7; --To determine the X-ray crystal structures of YgjD in complex with TCA, t 6 A and AMP.

2) Biophysical and biochemical characterization of

Cloning

The S. cerevisiae SUA5 gene was amplified from S. cerevisiae S288C strain (ATCC) genomic DNA by PCR using forward and reverse primers of 5'-AAAAAACCATGGGCTACCTTGGACGACATTTTTTGGCAAT-3' and 5'-TTTTTTCTCGAGTTAATGGTGATGGTGATGGTGAAACTGTATA-CAATTATTTGCAGCCGCCT-3', respectively. The gene contained a 6histidine tag DNA sequence at the 3'-end of and was sub-cloned into pET21d vector (Novagen) using the restriction sites NcoI (bold in forward primer sequence) and XhoI (bold in the reverse sequence). The gene-ligated vector was transformed into XL1 competent cells (Agilent technologies, Santa Clara, CA), followed by the streaking onto the argarose-2YT medium supplemented

with the ampicillin at a concentration of 100µg/ml. 3 colonies have been picked and individually inoculated into10 mL 2-YT medium supplemented with ampicillin at a concentration of 100µg/ml. The cells were gown overnight at 37 °C. The plasmids were extracted using QIAGEN Plasmid Mini Kit. Plasmids have been sequenced (Beckman Coulter Genomics) on the whole gene to check for the completeness of the ORF.

The plasmids for expression of the yeast KEOPS and subunits were previously constructed [START_REF] Collinet | Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project[END_REF]. Briefly, the polycistronic KEOPS sequence was chemically synthesized from S. cerevisiae KAE1, BUD32, CGI121, PCC1 and GON7 by DNA 2.0 (Menlo Park, USA) and was subcloned with an 6-histidine at C-terminus of Gon7 into an expression pUC derivative vector (pJ241_KEOPS/EKC-WT), which contains T7 promoter, LacI gene and a kanamycin resistance gene. In particular, each ORF of the five subunits has its own RBS (ribosome binding site), an AUG start codon as well as a stop codon. The ORFs of subcomplexes of Kae1-Bud32-6xHis and Bud32-Cgi121-6xHis have been amplified by PCR and inserted in a homemade polycistronic vector derived from pET28 giving rise to the plasmids 6-pK-Sc-Ka-Bu-a and 6-pK-Sc-Bu-Cg-a, respectively. Three other polycistronic vectors expressing Gon7-Pcc1-6xHis, Kae1-Bud32-Cgi121-6xHis and Gon7-Strep were chemically synthesized by genscript (Piscataway, USA) and subcloned into pET21a

vector which gave rise to the plasmids pET21a-BC1, pET21a-BC3 and pET21a-BC4, respectively.

The cloning of E. coli YgjD, YeaZ, YjeE and YrdC was carried out in Prof.

Forterre's group (IGM, Orsay) [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Shortly, E. coli ygjD, yeaZ, yjeE and yrdC were amplified by PCR from E. coli K-12 genomic DNA.

While ygjD, yeaZ and yrdC were subcloned into pET9a, pET9a and pET26b with an addition of 6xHis tags at the C-terminus, yjeE was subcloned into pET28a with an addition of Strep II tag (WSHPQFEK) at the C-terminus. The mutagenesis on E. coli ygjD, yeaZ, yjeE and yrdC genes was performed by

Bruno Collinet (This study in chapter 3). The following mutations have been generated by using the Quikchange mutagenesis kit (Agilent Technologies):

YgjD-F100E; YgjD-S97E; YgjD-S97R; YgjD-E12A; YgjD-V85E; YeaZ-Δ220;

YeaZ-R118A; YjeE-E108A; YjeE-T43A; YjeE-Y82A; YjeE-W109A; YrdC-R52A.

The cloning of P. abyssi Sua5 and P. abyssi Kae1 were carried out in Prof.

Forterre's group (Hecker et al, 2007b;[START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Shortly, the genes PAB1302 and PAB1159, which encode P. abyssi Sua5 and P. abyssi Kae1, were amplified by polymerase chain reaction (PCR) using total genomic DNA from the P. abyssi GE5 strain as template and cloned into pET26b and pET28a with an 6xHis tag added at the C-terminus, respectively.

Protein Expression and Purification

P. abyssi

Cloning and purification of E. coli tRNA Lys

The expression plasmid of pB-tRNA E.coli Lys(UUU) was obtained by subcloning the coding sequence of E. coli tRNA Lys(UUU) into a pBlue-Script vector, which contains a T5 promoter/lac operator sequence [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. The E.

coli tRNA Lys(UUU) was produced in XL1-Blue Competent Cells (Agilent Inc.)

transformed with plasmid pB-tRNA E.coli Lys(UUU) . The 10 ml transformant was precultured overnight at 37 °C and transferred into 800 ml auto-inducible Overnight Express Instant TB medium (Novagen), followed by growing for 20 hour at 37 °C. Cell pellets were prepared by centrifugation and washed with distilled water. Cells were lyzed and the total bulky RNAs were extracted with Trizol (Sigma) according to the manufacturer's procedure.

The coding sequences of P. abyssi tRNA Lys(UUU) , E. coli tRNA Ile(UCC) , S. The tRNAs after extraction from the cells or run-off transcription were applied to a 8M urea, 10% polyacrylamide gel for purification. After migration, the tRNA-containing slice as visualized by the UV shadowing (wavelength 254 nm) was excised and incubated with elution buffer (10 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA and 1% SDS) overnight at ambient temperature.

The tRNAs was precipitated from the eluate by 25% ethanol and 120 mM NaCl. The tRNAs were solubilized in RNase free H 2 O (Sigma) and diluted into buffer 25 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 and 0.2 mM EDTA. tRNAs were heated for 5 min at 95 °C and refolded by gradually cooling down. The Size-Exclusion Chromatography purification was also applied to isolate the homogenous and folded tRNA.

ATPase Assay

The ATPase activity (conversion into ADP and phosphate) was measured using a Lactate deshydrogenase-Pyruvate Kinase assay [START_REF] Tamura | Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine[END_REF]. The hydrolysis of ATP into ADP was coupled to the oxidation of NADH to NAD + and the consumption of NADH was determined by monitoring the absorbance at A 340 by a Cary WinUV 200 spectrophotometer (Agilent Inc.).

Assays were performed in a 10 mm cuvette at 18-22 °C in 150 µL assay buffer containing 50 mM HEPES pH 7.5, and an excess of pyruvate kinase and lactate dehydrogenase (Sigma). The absorbance was monitored until the reaction reaches the steady-state and recorded for 30 min. The reaction velocity was calculated by converting the decrease in the absorbance to consumption of NADH using an extinction coefficient of ɛ 340nm =6220 M -1 •cm -1 . ATPase reaction rates as a function of ATP concentration was fitted using Michaelis-Menten kinetics.

tRNA t 6 A Assay

The tRNAs were incubated with different proteins used in this study in assay buffer that comprised 50 mM Tris pH 7.4, 200 mM NaCl, 10 mM MgCl 2 , 25 mM KCl, 4 mM L-threonine, 2 mM ATP and 10 mM sodium bicarbonate.

The reactions were proceeded for 90 min at 37 °C and terminated by heating to 95 °C for 5 min, followed by fast cooling-down in ice water. Doing so, the tRNA was unfolded and the protein precipitates were removed by centrifugation. The tRNA was further enzymatically digested with nuclease P1, phosphodiesterase and alcaline phosphatase (Sigma), following the instructions of the commercial enzymes and a method developed for analysis of nucleosides by HPLC [START_REF] Gehrke | Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides[END_REF]. The digested nucleosides of tRNA were applied to HPLC using a µRPC C2/C18 ST 4.6/100 column (pore size 120 Å) and nucleosides were detected by UV at 258 nm. The column was equilibrat-ed with buffer A (10 mM Ammonium Acetate, pH 7.0). The gradient used for elution of was 100% buffer A from 0 to 8 min followed by a linear gradient of 0-100% buffer B (6 mM Ammonium Acetate, pH 7.0 and 40% acetonitrile) from 8 to 30 min. The standard Cytosine (C), Uracil (U), Guanine (G), Adenine (A) (Sigma) and t 6 A (kindly provided by R. W. Adamiak, Polish Academy of Sciences) were used as reference for determination of the elution time of each digested nucleosides in the reaction mixture [START_REF] Adamiak | The chemical synthesis of the anticodon loop of an eukaryotic initiator tRNA containing the hypermodified nucleoside N6-/N-threonylcarbonyl/-adenosine/t6A/1[END_REF]. The area under each peak that corresponds to C, U, G, A and t 6 A have been calculated

and used for quantification of the digested nucleosides of tRNA. The percentage of t 6 A is calculated as the average of area of t 6 A to area of C, U and G, respectively, normalized to tRNA alone and tRNA modified by wild-type proteins. MS analyses of nucleosides were performed by mass spectrometer Micro TofQ (BRUKER DALTONICS) equipped with HPLC (Ultimate3000, DI-ONEX) using the µRPC C2/C18 ST 4.6/100 column. MS was employed to confirm the masses of C, U, G, A and t 6 A from digested nucleosides.

Multi-Angle Laser Light Scattering

The characterization of the olimerization state and association of proteins was performed with the SEC-TDA equipment (Malvern, France). The Superdex TM 200 (75) HR 10/30 columns (GE Healthcare) or Bio-SEC3 column (Agilent) for Size-Exclusion Chromatography was equilibrated with respective buffer. 80-100 µl protein samples at a concentration of 3-5 mg/ml were injected at a flow rate of 0.5 ml min -1 onto the SEC column and eluted with the equilibration buffer at a flow rate of 0.3 ml•min -1 . Elution was followed by a UV-

Isothermal Titration Calorimetry

Proteins and/or ligands were solubilized in appropriate homogenous buffer.

Protein concentrations determined spectrophotometrically at 280 nm using their extinction coefficients. All experiments were performed on an iTC200 system (MicroCal ® ) at 20℃, 25 ℃ or 35 ℃. The cell was filled with 300 µl of the protein samples and titrated with 2.0 µl injections. A first injection of 0.4 µl was followed by 20 injections of 2 µl at intervals of 180 s. The data were analyzed according to the one binding-site model using the MicroCal Origin software provided by the manufacturer. The plots were fitted using the Origin software according to a one-site binding model or other binding models.

Crystallization and Data Collection

Purified proteins were applied for either crystallization or cocrystallization with their binding ligands, using a vapor-diffusion method. The initial screen of crystallization conditions was performed using commerical crystallization Glycerol or PEG400 up to 30% and then flash-cooled and stored in liquid nitrogen. Crystals were tested for diffraction at beamlines PX1 and PX2 at SO-LEIL Synchrotron (Saint-Aubin, France) and ID23-1 and ID23-2 at ESRF (Grenoble). Native diffraction datasets and single-wavelength anomalous dispersion (SAD) at Se-edge were collected using Pilatus detectors at certain wavelengths. Both native and SAD datasets were processed by XDS package and converted to mtz reflection file format [START_REF] Kabsch | Xds[END_REF] or processed by Mosflm [START_REF] Battye | iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM[END_REF].

X-ray Structural Determination and Model Refinement

The initial models were obtained against the processed mtz reflection files by molecular replacement performed with MOLREP [START_REF] Vagin | Molecular replacement with MOLREP[END_REF] and PHASER MR [START_REF] Mccoy | Solving structures of protein complexes by molecular replacement with Phaser[END_REF]. The crystal structures of the related were used as search models, of which certain loop regions and residue side chains were removed by using the Chainsaw program in CCP4 package [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF]. The models from the initial molecular replacement were visually checked for validity by using COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]) and further completed using PHENIX AutoBuild [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF][START_REF] Terwilliger | Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard[END_REF]. The models were refined against the diffraction data by using REF-

MAC5 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] and PHENIX Refine [START_REF] Afonine | Towards automated crystallographic structure refinement with phenix.refine[END_REF]. The final models were completed and examined manually by COOT, followed by the analysis of the electron density fit. Validation used the Ramachandran plot option in COOT and the PROCHECK program [START_REF] Laskowski | PROCHECKa program to check the stereochemical quality of protein structures[END_REF]. The determination of the structure from the dataset collected by SAD was initially carried out by SHELXD [START_REF] Schneider | Substructure solution with SHELXD[END_REF], which was used to identify and locate the selenium sites. The Phaser SAD Pipeline (Read & McCoy, 2011) and Buccaneer [START_REF] Cowtan | The Buccaneer software for automated model building. 1. Tracing protein chains[END_REF] were used to build the model.

The final model building and refinement were performed in a same way as the native structures.

Small-Angle X-ray Scattering (SAXS)

SAXS experiments were carried out on the SWING beamline at the SO-LEIL synchrotron radiation facility (Saint-Aubin, France). The sample to detector (Aviex CCD) distance was set to 1500 mm, allowing reliable data collection over the momentum transfer range 0.01 Å -1 < q < 0.6 Å -1 with q=4πsinθ/λ where 2θ is the scattering angle and λ the wavelength of the X-rays (λ= 1.0 Å). The solution was injected into the SAXS flow-through capillary cell (1.5 mm in diameter) under vacuum. In order to use complexes solutions fully devoid of isolated partners, SAXS data were collected directly after elution through an online size-exclusion high-performance liquid chromatography (Bio-SEC3 Agilent) at a flow rate of 200 µL/min. SAXS data were collected online continuously, with a frame duration of 1 s and a dead time between frames of 0.5 s. The first 60 frames collected during the first minutes of the elution flow were averaged to account for buffer scattering. The 20 frames corresponding to the top of the elution peak were averaged and used for data processing after baseline subtraction. SAXS data were normalized to the intensity of the incident beam and background (i.e. the elution buffer) subtracted using the programs FoxTrot (courtesy of SWING beamline) and Primus [START_REF] Konarev | ATSAS 2.1, a program package for small-angle scattering data analysis[END_REF][START_REF] Konarev | PRIMUS: a Windows PC-based system for small-angle scattering data analysis[END_REF]. The scattered intensities were displayed on an absolute scale using the scattering by water. The molar mass also was obtained independently from the whole I(q) curve using the Porod volume and the program DATPOROD developed by Svergun's team (ATSAS package at http://www.embl-hamburg.de/biosaxs/software.html). The ab initio envelope of the scattering object was determined using the program GASBOR [START_REF] Svergun | Determination of domain structure of proteins from X-ray solution scattering[END_REF], which describes it as a chain of dummy residues. And ab initio model was used to compare with the crystal structure for determining the oligomeric states of the proteins. Scattering patterns from crystal structures were calculated using the program CRYSOL [START_REF] Svergun | CRYSOL -a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates[END_REF] and then were adjusted to the experimental curve. During this procedure the Histag and missing residues absent in the crystal structure, were added. The quality of the fits was characterized by the parameter:

where N is the number of experimental points, c is a scaling factor, and I calc (q j ) and σ(q j ) are the calculated intensity and the experimental error at the scattering vector q j .

Other Biophysical Techniques

Atomic Absorption Spectroscopy (AAS) was used to determine the metals bound in Pa-Kae1, Sc-Kae1, Qri7 and YgjD. Proteins were dialyzed overnight against their respective purification buffers supplemented with 0.1 mM of EDTA. The dialyzed protein samples, dialysis buffers untreated proteins were applied to Varian SpectrAA 220 spectrometer (Varian, Inc). Samples was chromatographed on to a TSK3000SWXL column (Tosohaas, Japan, 7.8 × 300 mm 2 ) operated at 0.5 ml/min in the same buffer. The reference absorbance and values were obtained by applying the MnCL 2 and ZnCl 2 in the corresponding buffers. The values were deduced to calculate the binding stoichiometry of metal to proteins, whose concentrations were determined by NanoDrop (Thermo Scientific™).

The NMR 2D HSQC experiment was carried out at 25 ℃ on a Bruker Avance 600 MHz spectrometer for analysis of the Gon7 folding status in solution. The residue-specific { 1 H}-15 N heteronuclear NOE was measured by dividing the peak intensity in two 2D spectra recorded with and without a 3 s proton presaturation achieved by 120 ° 1 H pulses following a 5 s delay. The 15 Nlabled Gon7 was prepared in phosphate buffer and used at a concentration of 2 mg/ml.

Figures and graphics

All structural figures were generated with PyMol (Molecular Graphics System, Schrödinger, LLC). The projection of the sequence conservation onto the structure was performed by CONSURF [START_REF] Landau | ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures[END_REF]. The representation of the electrostatic surface of protein was created by PDB2PQR [START_REF] Dolinsky | PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations[END_REF].

Introduction

Sua5/YrdC family proteins are present in the three domains of life (Galperin & Koonin, 2004a;[START_REF] Klima | A putative helicase, the SUA5, PMR1, tRNALys1 genes and four open reading frames have been detected in the DNA sequence of an 8.8 kb fragment of the left arm of chromosome VII of Saccharomyces cerevisiae[END_REF][START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. Phylogenetic distribution shows that YrdC may either exist as an independent protein or as a fusion protein to other domains or proteins. For example, YrdC from bacteria and human exists as an independent protein [START_REF] Chen | Isolation and identification of a novel cDNA that encodes human yrdC protein[END_REF][START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF], but in the Sua5 proteins from yeast and archaea it is combined with a so-called Sua5 domain [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF][START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF]. In E. coli, YciO shares 29% sequence identity with YrdC and is sometimes included as a member of Sua/YrdC protein family [START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. Both S. cerevisiae Sua5 (Sc-Sua5) and E. coli YrdC (Ec-YrdC) are essential for cell life [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Na | Isolation and characterization of SUA5, a novel gene required for normal growth in Saccharomyces cerevisiae[END_REF] but their function remained longtime unknown (Galperin & Koonin, 2004a). YrdC/Sua5 was reported to bind nu- In the bacterial HypF and TobZ proteins, a YrdC-like domain is fused with a Kae1like domain [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF][START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF]. TobZ is involved in the synthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF] and HypF in the maturation of active [NiFe] hydrogenases [START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF]. In both TobZ and HypF, the YrdC domains utilize ATP and carbamoyl phosphate to synthesize an intermediate carbamoyladenylate (CA) whose carbamoyl-moiety is subsequently transferred onto acceptor tobramycin and onto Cterminal cysteine of HypE by Kae1-domains in TobZ and HypF, respectively [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF][START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF].

The t 6 A modified base is present 3' adjacent to the anticodon of tRNAs that recognize the ANN-codons [START_REF] Chheda | Isolation and characterization of a novel nucleoside, N-[(9beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]threonine, from human urine[END_REF][START_REF] Takemura | The primary structure of isoleucine transfer ribonucleic acid from Torulopsis utilis. Complete digestion with ribonucleases and construction of model of its secondary structure[END_REF]. t 6 A plays an essential role in translational fidelity through disabling the intramolecular Watson-Crick pairing of A 37 with U 34 and enhancing the codon-anticodon pairing [START_REF] Fvt | The role of modifications in codon discrimination by tRNA(Lys)UUU[END_REF][START_REF] Parthasarathy | Conformation and possible role of hypermodified nucleosides adjacent to 3'-end of anticodon in tRNA: N-(purin-6-ylcarbamoyl)-L-threonine riboside[END_REF]. The biosynthesis of tRNA t 6 A requires tRNA, ATP, L-threonine and bicarbonate as substrates [START_REF] Elkins | The enzymatic synthesis of N-(purin-6ylcarbamoyl)threonine, an anticodon-adjacent base in transfer ribonucleic acid[END_REF]. The reaction is carried out by YrdC, YgjD/YeaZ/YjeE in bacteria [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF].

Kae1 is part of an ancient protein complex KEOPS that is further composed of Bud32, Cgi121 and Pcc1 in archaea and a fifth subunit, Gon7, in yeast [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]Kisseleva-Romanova et al, 2006b;[START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Interestingly, yeast mitochondrial t 6 A synthesis only requires Qri7 and Sua5 [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF] The crystal structure of Ec-YrdC is made of an α/β twisted mixed open-sheet structure. The 7 α-helices and 7 β-strands create an unusual topology of YrdC consisting of three parts: the first is composed of α1, β1, β2 and α2; the second comprises β3, α3, α4, β3, α5 and β5; and the third contains α6 and β6. The C-terminal α7 and β7 folds down and spans over the remainder of the domain [START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. The structures of YrdC and YciO are very closely related, with an RMSD of 1.52 Å over 189 residues [START_REF] Jia | Crystal structure of the YciO protein from Escherichia coli[END_REF]. The most striking structural feature of Ec-YrdC is a large concave surface formed by β3, β4, β5, β7, and α5. The electrostatic potential of this depression concave surface is positive, providing a support for the binding of double-strand RNA or tRNA to Ec-YrdC [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF]Harris et al, 2011;[START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF]. The crystal structure of S. tokodaii Sua5 (St-Sua5) revealed that its N-terminal YrdC domain is identical to the structure of Ec-YrdC (RMSD of 2.4 Å for a sequence similarity of 42.9% over 213 residues) [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF]. The C-terminal Sua5 domain of St-Sua5 consists of 3 α-helices and 5 βstrands and adopts a classic Rossmann fold [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF]. Interestingly, the Sua5 domain is structurally close to the M. jannaschii HypB and T. aquaticus Fth proteins, both of which are GTP-binding proteins [START_REF] Freymann | Functional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP[END_REF][START_REF] Gasper | Structural insights into HypB, a GTP-binding protein that regulates metal binding[END_REF]. The St-Sua5 structures show that the active site is accommodated by the YrdC domain and is in proximity to the Sua5 domain. The linker between the two domains was disordered in the crystal structures but probably contributes to the formation of ATP-binding site. In the active site of St-Sua5, the phosphates-moiety of AMPPNP interacts with a highly conserved KRSN tetrad [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF]. The mutations on the KRSN tetrad of Sc-Sua5 not only abolished its activity in biosynthesis of t 6 A [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF] but also abrogated telomeric DNA binding [START_REF] Meng | Sua5p a single-stranded telomeric DNA-binding protein facilitates telomere replication[END_REF]. The KRSN tetrad is not conserved in YciO, likely accounting for its incapability in catalyzing the biosynthesis of tRNA t 6 A [START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Jia | Crystal structure of the YciO protein from Escherichia coli[END_REF].

The 20-residue loop linking the YrdC and Sua5 domains was not observed in the reported crystal structures of St-Sua5. Since this loop spans the active site pocket, it may contribute to substrate binding and activity. In the present study we report a 2.6

Å crystal structure of full-length P. abyssi Sua5 (Pa-Sua5) in complex with Lthreonine and pyrophosphate. The structure provides an invaluable information for the ATP-pyrophosphatase activity of Sua5. Furthermore, this structure revealed for the first time a 20-residue regulatory linker between YrdC domain and Sua5 domain.

Binding experiments with ligands have been also characterized.

Materials and methods

Expression and purification of the recombinant Pa-Sua5

The PAB1032 (gi|5459014) gene encoding Pa-Sua5 was cloned into the pET26b vector with addition of a 6xHis tag at the C-terminus [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF] and was heterologously overexpressed in E. coli Rosetta pLysS (DE3) cells. The induction of the overexpression of Pa-Sua5 was initiated by adding IPTG to a final concentration of 0.5 mM for 3 hours at 37 °C when the OD 600 of the cell culture reached 0.6. The cell pellets were prepared by centrifugation at 6000 g for 15 min at 25 °C and suspended and stored in buffer A (20 mM Tris pH 7.5, 200 mM NaCl and 5 mM 2-mercaptoethanol) at -20 °C. The cell pellets were defrozen at 37 °C in a water bath and lysed by sonication on ice, followed by incubation at 65 °C for 30 min. The cell lysis was centrifuged at 20,000 g for 30 min and the supernatant was applied to an Immobilized metal ion affinity chromatography (IMAC) that was packed with 3 ml Nikel Resin (Qiagen) and equilibrated with buffer A. The resin bound with proteins in the column was washed with 30 ml buffer A and proteins were eluted gradually with buffer A supplemented with imidazole at a concentration ranging from 100 mM to 400 mM and analyzed by SDS-PAGE. The elution fractions containing PaSua5 were loaded on a S75 16/60 gel filtration column (GE health), equilibrated in buffer A. The Pa-Sua5 fractions above 95% purity (SDS-PAGE) were pooled and concentrated to 15.0 mg/ml.

Calorimetric measurement by Isothermal Titration Calorimetry (ITC)

Samples were solubilized either in buffer A or in a phosphate buffer (30 mM phosphate-K pH 6.8, 200 mM NaCl and 5mM 2-mercaptoethanol). Protein concentrations determined spectrophotometrically at 280 nm using extinction coefficient of 28,420 M -1 cm -1 . All experiments were performed on an iTC200 system (MicroCal) at 35 ℃ or in some cases at 25 ℃. The cell was filled with 300 µl of the protein samples and titrated with 2 µl injections that containing ligand at a concentration 10-12 folds of the protein in the cell. A first injection of 0.4 µl was followed by 20 injections of 2 µl at intervals of 180 seconds. The plots were fitted using the MicroCal Origin software according to a one-site binding model.

Size-Exclusion-Chromatography coupled Multi-Angle Laser Light Scattering (SEC-MALLS)

The molecular weight of Pa-Sua5 was determined using SEC-MALLS with the SEC-TDA equipment (Malvern, France). The Superdex TM 200 (75) HR 10/30 columns (GE Healthcare) or Bio-SEC3 column (Agilent) for Size-Exclusion Chromatography (SEC) was equilibrated with buffer A that was used in the purification step. 80 µl protein Pa-Sua5 at a concentration of 5 mg/ml was injected at a flow rate of 0.5 ml min -1 onto the SEC column and eluted with the equilibration buffer at a flow rate of 0.3 ml min -1 . Elution was followed by a UV-visible spectrophotometer, a differential refractometer, a 7° Low Angle Light Scattering detector, a 90° right angle light scat-tering detector and a differential pressure viscometer. The OmniSEC software program was used for the acquisition and analysis of the Viscotek data. BSA was used as standard reference protein of known molecular weight, concentration, refractive index increment (dn/dc=0.185 ml•g -1 ), and intrinsic viscosity to determine the instrument response factors of the detectors for the mobile phase being used.

Crystallization, data acquisition and structure determination

Pa-Sua5 at a final concentration of 9.0 mg/ml was either crystallized alone or cocrystallized with ATP/MgCl 2 /L-threonine/bicarbonate (all ligands were supplemented at 5.0 mM) by the sitting-drop vapor diffusion method. Crystals were obtained within 3 days from a crystallization reservoir containing 0.1 M Tris pH 8.5-8.8, 3.5M ammonium sulfate. Crystals were cryo-protected in crystallization reservoir solution supplemented with 30% glycerol and flash-frozen in liquid nitrogen. The X-ray diffraction data were collected at 100K at the Soleil Synchrotron Light Source Proxima 1 (wavelength 0.98011 Å, PILATUS 6M detector). The images were successfully processed and scaled with the XDS package [START_REF] Kabsch | Xds[END_REF]. The Ramachandran plot analyses of the model by PROCHECK [START_REF] Laskowski | PROCHECKa program to check the stereochemical quality of protein structures[END_REF] revealed 4 residues as outliers in the asymmetric unit. However, the electron density for these 4 residues was unambiguously defined. The data processing and refinement statistics are summarized in table 1.

The structure was solved by molecular replacement with the program MOLREP implemented in the CCP4 suite [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] using the structure of St-Sua5 as the search model (PDB code: 2EQA). The model building was carried out with program Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and the structure refinement was performed with Refmac5 [START_REF] Winn | Macromolecular TLS refinement in REFMAC at moderate resolutions[END_REF]. There are 4 Pa-Sua5 monomers in the asymmetric unit. The electron density is well defined for all residues between 2-339 as well as several histidines tagged at the C-terminus in the asymmetric unit.

Sequence Alignment and structure comparison

The sequence conservation was analyzed using the program ConSurf [START_REF] Armon | ConSurf: an algorithmic tool for the identification of functional regions in proteins by surface mapping of phylogenetic information[END_REF] with the structure of Pa-Sua5 as the query structure. Based on the default criteria, the program ConSurf employed CS-BLAST to identify 499 hits and performed the calculation on 150 homologous sequences with the lowest E-value. The alignment truncated the sequences of Pa-Sua5 and classified them into YrdC-like domain and Sua5-like domain. The frequency of the occurrence of amino acids on the consensus sequence was produced by WebLogo [START_REF] Crooks | WebLogo: a sequence logo generator[END_REF]. The structural comparison was performed with SSM [START_REF] Krissinel | Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions[END_REF]

Results

Overview of the structure of Pa-Sua5

We first crystallized Pa-Sua5 alone and determined its structure at 2.8 Å. The asymmetric unit (AU) contains four identical copies of Pa-Sua5 (average root-meansquare-deviation (RMSD) of 0.6 Å over 341 Cα atoms). Sec-Malls measurements indicate Pa-Sua5 is present in solution as a monomer (Fig. S4A). Pa-Sua5 consists of a YrdC-and a Sua5-domain, shown in blue and yellow, respectively in Fig. 1A.

The overall structure of Pa-Sua5 is identical to that of S. tokodaii Sua5 (St-Sua5)

with an RMSD of 0.67 Å over 318 Cα atoms (Fig. 3A). A residual electron density cloud at the bottom of the catalytic pocket could be fitted by a L-threonine molecule, which co-purified with the enzyme. L-threonine forms hydrogen bonds with tfour highly conserved residues Thr 35 , Arg 121 , Ser 181 and Arg 195 (Fig. 2A). The L-threonine overlaps with the threonyl-moiety of the threonylcarbamoyladenylate (TCA) bound to

St-Sua5 (Fig. 3).

In order to map the substrate/product binding sites of Pa-Sua5, we co-crystallized

Pa-Sua5 with ATP, L-threonine, bicarbonate and MgCl 2 . Crystals were obtained in the same conditions as for the L-threonine bound form and diffracted at 2.6 Å. There was no indication for bound nucleotide but we observed residual electron density in the active site cavity, which could be satisfactorily fitted by a pyrophosphate ion (Fig. 1A and Fig. S1). The pyrophosphate ion displaces two well-bound water molecules present in the L-threonine bound structure (Fig. S1). Pyrophosphate was likely generated by the hydrolysis of ATP into AMP during the crystallization. L-threonine sits at the bottom and pyrophosphate atop of the catalytic pocket of the YrdC domain, in proximity to the linker peptide (Fig. 1 andFig3). The pyrophosphate ion occupies a positively charged electrostatic surface potential patch (Fig. 1C). The position of the pyrophosphate superposes to that of the β-and γ-phosphates of the AMPPNP in St-Sua5 (Fig. 3C). The pyrophosphate oxygens are coordinated by 9 hydrogen bonds with residues Lys 56 , Arg 58 , Asn 145 , Ser 147 and His 234 all of which, except His 234 , are totally conserved (Fig. 2B and Fig. 1). Interestingly, some of the linker residues contribute to the coordination of the pyrophosphate (Fig. 2B). At the top of the active site is an opening that could serve as a path for release of the pyrophosphate (Fig. S1E). ). It was shown that the mutation of either Lys 93 or Arg 95 of Sc-Sua5 (corresponding to Lys 56 and Arg 58 of Pa-Sua5, respectively) are inactive in t 6 A biosynthesis [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The main structural difference between Pa-Sua5 and St-Sua5 resides in the linker region between the YrdC and Sua5 domains (residues from Ile 213 to Tyr 232 ; Fig. 3, shown in red). This linker region was disordered in the St-Sua5 structures but has well defined electron density in Pa-Sua5 (Fig. 3A). The linker starts as a short α-helix ( 215 PAVY 218 ) and continues by a long U-turn (Fig. 3A-C) that plunges into the active site pocket. It stabilizes the bound pyrophosphate ion (Fig. S1D) through hydrogen bonds with Gly 229 and His 234 (Fig. 2B). The position of the linker overlaps completely with the AMPPNP or TCA molecules bound to St-Sua5 (Fig. 3A and4C) [START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF][START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF].

The Binding of L-threonine and Pyrophosphate to Pa-Sua5

We then quantified the binding of L-threonine, AMPCPP, pyrophosphate, bicarbonate and AMP to Pa-Sua5 by ITC. We failed to measure any heat exchange during the titration of Pa-Sua5 with L-threonine (Fig. S2). Since we observed Lthreonine in the crystal structure of the protein alone, this compound probably remains bound during the purification process. ATP is hydrolyzed by Pa-Sua5 into AMP in presence of L-threonine [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF], we measured the binding of the non-hydrolyzable AMPCPP and obtained a Kd value of 2.1 µM (Fig. 2C). The titration of Pa-Sua5 with pyrophosphate yielded Kd value of 50.0 µM (Fig. 2D). By contrast, no heat exchange was observed upon titration of Pa-Sua5 with AMP nor bicarbonate (Fig. S3).

Discussion

The tRNA t 6 A modification proceeds through the biosynthesis of an unstable TCA

Conclusion

We report the crystal structure of P. abyssi Sua5 in complex with L-threonine and pyrophosphate generated by hydrolysis of ATP. We identified a gating loop that links the YrdC-and Sua5-domains and propose that this linker may cover the active site during the synthesis of TCA to stabilize intermediates formed during the reaction cycle.

CHAPTER ǁ‖

The structural model of yeast KEOPS

Introduction

Telomeres are the physical ends of linear chromosomes and they shorten along with genome replication during cell division. Telomere capping in yeast is carried out by a protein complex containing Cdc13, Stn1 and Ten1 [START_REF] Martin | Protection of telomeres by a conserved Stn1-Ten1 complex[END_REF][START_REF] Pennock | Cdc13 delivers separate complexes to the telomere for end protection and replication[END_REF]. In a search for new S. cerevisiae genes involved in capping, CGI121 was identified from a genome-wide study of a nonessential gene knock-out library that could suppress a temperature sensitivity allele cdc13-1 [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]. Cgi121 is part of a multiprotein complex, dubbed KEOPS, for Kinase, Endopeptidase and Other proteins of Small size.

The yeast KEOPS complex is composed of five proteins: Gon7, Pcc1, Kae1, Bud32 and Cgi121, and was shown to promote both telomere uncapping and elongation [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. In a parallel unrelated study, Kisseleva-Romanova et al. identified the same complex and documented its role in transcriptional regulation of GAL genes in yeast (Kisseleva-Romanova et al, 2006a). Interaction of the KEOPS complex with chromatin suggested that it could also function as a transcription factor (Kisseleva- et al, 2006a). Kae1 belongs to an universally conserved Kae1/Qri7/YgjD protein family (Galperin & Koonin, 2004b) whose members are implicated in telomere homeostasis [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF], genome stability [START_REF] Oberto | Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance[END_REF] and tRNA N 6 -threonylcarbamoyladenosine (t 6 A) modification [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF].

Romanova

The t 6 A is universally present at position 37 of tRNAs that recognize ANNcodons [START_REF] Parthasarathy | Conformation and possible role of hypermodified nucleosides adjacent to 3'-end of anticodon in tRNA: N-(purin-6-ylcarbamoyl)-L-threonine riboside[END_REF][START_REF] Takemura | The primary structure of isoleucine transfer ribonucleic acid from Torulopsis utilis. Complete digestion with ribonucleases and construction of model of its secondary structure[END_REF]. tRNA t 6 A enhances the codon anti-codon interaction at the first AUG codon and is important for maintaining the translation fidelity [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF][START_REF] Fvt | The role of modifications in codon discrimination by tRNA(Lys)UUU[END_REF].

Abolition of the t 6 A modification therefore affects the translation of many proteins and hence might indirectly affect multiple cellular processes (Daugeron et al, 2011a;[START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. Comparative genomics and biochemical experiments showed that KEOPS and Sua5, of which the later belongs to the universal and essential Sua5/YrdC protein family, are necessary and sufficient for the biosynthesis of tRNA t 6 A [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Qri7 is the yeast mitochondrial paralog of Kae1 and is capable of compensating for the lack of Kae1 in the t 6 A biosynthesis (El Yacoubi et al, 2011). Recent study showed that Sua5 and Qri7 constitute the minimal system for the in vitro t 6 A biosynthesis [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. In bacteria, the in vitro t 6 A biosynthesis requires YrdC and YgjD/YeaZ/YjeE [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. In t 6 A biosynthesis reaction in archaea, Sua5

synthesizes an unstable intermediate threonylcarbamoyladenylate (TCA), of which the threonylcarbamoyl-moiety is transferred onto A 37 of tRNA by KEOPS [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. While the catalytic center is located in Kae1, the functions of other KEOPS subunits remain largely unclear, so is that for yeast KEOPS.

The crystal structures of archaean Kae1 (Hecker et al, 2007b), mitochondrial Qri7 [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF] and bacterial YgjD [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF] showed that they belong to the ASKHA (acetate and sugar kinases/Hsp70/actin) superfamily [START_REF] Buss | Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases[END_REF]. A non-heme Fe(III) was found in the active site of P. abyssi Kae1, liganded by two histidines and one aspartate, which form a conserved metal-binding motif in Kae1/Qri7/YgjD (El Yacoubi et al, 2011). The metal directly contacts the γ-and -β phosphates of the AMPPNP in archaean Kae1 (Hecker et al, 2007a;[START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF] and the ATPγS in S. typhimurium YgjD [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. The crystal structures of archaean Pcc1, Kae1, Bud32 and Cgi121 as well as some KEOPS subcomplexes have been determined [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The ensemble of structures of subcomplexes allowed reconstructing a structural model of KEOPS from archaea [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. KEOPS displays a linear rearrangement of subunits, with Pcc1 at one end, Kae1 and Bud32 in the middle and Cgi121 at the other end. N-terminal domain of Kae1 binds to Pcc1 and its C-terminal domain protrudes into the two lobes of Bud32, which is structurally related to the small-size atypical RIO kinases (LaRonde-LeBlanc & Wlodawer, 2005a).

Cgi121 binds to the N-terminal lobe of Bud32 and presently, has no structural analogs. The functional importance of the protein-protein interactions that mediate the assembly of S. cerevisiae KEOPS was tested in vivo. Mutations that lead to disruption of the Kae1/Bud32 and Pcc1/Kae1 complexes had pleiotropic phenotypes including slow growth, telomere shortening, defective transcriptional regulation, similar to effects caused by deletion of either KAE1 gene or BUD32 gene (Daugeron et al, 2011b;[START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Disruption of the Cgi121/Bud32 also caused slow growth and short telomeres but the effect was milder than for Kae1/Bud32 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

Archaean Pcc1 functions as a dimerization module that further mediates the formation of a dimer of heterotetrameric KEOPS, exhibiting a V-shape molecular architecture in which Pcc1 dimer is situated at the base connecting the two arms [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

Finally, the fifth component of KEOPS complex from yeast is Gon7 for which no homologues have been found outside the fungal lineage. Very little is known about its specific function, though it's required for t 6 A biosynthesis and telomere length maintenance [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF]. Gon7 gene expression can be induced during osmotic stress (Garay- et al, 2000) and the Gon7-knockout yeast strain shows a defect in mannosyl phosphorylation of mannoprotein-linked oligosaccharides [START_REF] Corbacho | A genome-wide screen for Saccharomyces cerevisiae nonessential genes involved in mannosyl phosphate transfer to mannoprotein-linked oligosaccharides[END_REF]. We determined the crystal structure of the Gon7/Pcc1 complex and investigated the role of Gon7 within the KEOPS complex. We show that Gon7 interacts with Pcc1 and binds to Pcc1 at the opposite site of that between Pcc1 and Kae1, mimicking the second Pcc1 subunit as in the archaean Pcc1 homodimer. We further determined the structure of the yeast Cgi121/Bud32 subcomplex and construct a 3D model for the complete yeast KEOPS complex. This model revealed the presence of a deep conserved crater leading to the active site of Kae1 and surrounded by residues from Gon7, Pcc1, Kae1 and Bud32.

Arroyo

Materials and methods

Cloning, expression, and purification of yeast KEOPS complex and the subcomplexes

We ordered a 3.5 kb DNA synthetic fragment that contains five ORFs of S.

cerevisiae KEOPS subunits and subcloned it into a pUC backbone vector by DNA2.0 (https://www.dna20.com/). In this expression vector named pJ241-KEOPS, the five ORFs of KEOPS subunits have been placed in the following order from the 5' end to the 3' end: yKae1; yBud32; yCgi121; yPcc1 and yGon7-6xHis [START_REF] Collinet | Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project[END_REF]. Every ORF possesses its own RBS and

Stop codon and only one T7 promotor and one terminator sequence has been added respectively at the 5' and 3' end of the synthetic gene. The cloning of Bud32-Cgi121 complex was carried out using the same strategy as for KEOPS complex. Briefly, the new expression vector named 6-pK-Sc-Bu,Cg-a contains the upstream BUD32 and downstream CGI121 with 6 extra histidine codons at its 3' end. In short, two other polycistronic vectors expressing Gon7-Pcc1-6xHis and Gon7-strep were chemically synthesized by genscript (Piscataway, USA) and subcloned into pET21a vector which gave rise to the plasmids pET21a-BC1 and pET21a-BC4, respectively.

KEOPS-his and Bud32-Cgi121-his were heterologously produced E. coli BL21 AI cells (Invitrogen) and E. coli BL21 Rosetta pLysS (Novagen) transformed with pUC-KEOPS and 6-pK-Sc-Bu,Cg-a, respectively. The expression of KEOPS was induced by adding 0.2 % of arabinose and IPTG to a final concentration of 0.5 mM, followed by growing the culture overnight at 15 °C. The expression of Bud32-Cgi121 was induced by adding IPTG to a final concentration of 0.5 mM for 3.5 Hour at 37 °C. native and SeMet-substituted Gon7-Pcc1-6xHis, and N 15 -labeled Gon7-strep were produced in E. coli RosettaBlue (DE3) pLysS Cells (Novagen) transformed with pET21a-BC1 and pET21a-BC4, respectively. The expression was induced by adding IPTG to a final concentration of 0.5 mM, followed by growing the cell overnight at 15 °C.

Cells were harvested by centrifugation and resuspended in 30 mL of lysis buffer (Tris 20 mM pH 7.5, NaCl 200 mM and 2-mercaptoethanol 5 mM) and either stored at -20 °C or directly used for proteins purification. Prior to purification, cells were lysed by sonication (branson sonifier 250) and centrifuged at 20,000g for 30 minutes. For KEOPS-His, Bud32-Cgi121-His, Gon7-Pcc1-His, the supernatant (soluble fraction) was then applied on Immobilized Metal Affinity Chromatography (IMAC) using Ni-IDA resin (Macherey Nagel). After following a washing step with 25-30 mL of lysis buffer, elution of the five protein was performed by loading successive fractions of 1mL of lysis buffer supplemented with increasing concentrations of imidazole. A second sizeexclusion chromatography step was performed on Superdex S200 (for KEOPS-His) or Superdex S75 column (GE-Healthcare). In some cases, an extra Mono Q anion-exchange purification step was applied. Fractions containing proteins with satisfactory purity were pooled and prepared by concentration, followed by flash freezing and stored at -80°C or directly used for subsequent experiments. The purification of N 15 -labeled Gon7-Strep was carried out with Immobilized Metal Affinity Chromatography (IMAC) using Strep-Tactin resin resin (IBA), according to the manufacturer's instructions. A final sizeexclusion chromatography step was performed on Superdex S75. Fractions contained Gon7-Strep was pooled and prepared for immediate use.

Crystallization and resolution of the structures

Bud32-Cgi121 complex

Bud32-Cgi121-His at a concentration of 10 mg/ml was used for crystallization using a method of vapor diffusion with hanging drops. Crystals were obtained at 20 °C from crystallization solution that contains Bud32-Cgi121-His at 5 mg/ml 50 mM and Tri-sodium pH 5.60, 10% 2-Propanol and 10% PEG4000

or 50 mM Sodium Acetate pH 4.6, 1.0 M Ammonium sulfate. The crystals were cryo-protected by transfer into the crystallization solution with increasing glycerol concentrations up to 30% v/v and then flash cooled in liquid nitrogen.

The diffraction dataset was collected at SOLEIL Synchrotron beamline Proxima 1 (Saint Aubin, France). Data were processed using the XDS package [START_REF] Kabsch | Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants[END_REF]. The space group was I4 1 with one heterodimer per asymmetric unit. The structure was determined by the molecular replacement method using the program MOLREP [START_REF] Vagin | An approach to multi-copy search in molecular replacement[END_REF], using M. jannaschii

Cgi121 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF] and Bud32 (Hecker et al, 2007a) as search models.

The model was further improved by the PHENIX AutoBuild wizard [START_REF] Terwilliger | Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard[END_REF]. This model was then refined against the 2.25 Å resolution native dataset using REFMAC5 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] and then manually built with COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]). The final model for the Cgi121-Bud32-His contain residues from 3 to 57 and 71 to 258 from Bud32 as well as residues 1 to 179 from Cgi121. We also observed well defined electron densities for two glycerol, two acetate and one adenosine molecule. In parallel, we cocrystalized Bud32-Cgi121-His with various nucleotide and obtained crystals Bud32-Cgi121 in complex with MgCl 2 and AMPPNP, or ATP, or AMPCPP, or ADP.

The crystals were grown under the same conditions as for the Bud32-Cgi121 apo-complex. These crystals belong to the same I4 1 space group and diffracted up to 1.8 Å resolution on SoLEIL Synchrotron beamline Proxima 1 (Saint Aubin, France). Analysis of the Fourier difference map enabled us to clearly locate all the ADP-moieties of nucleotides that are bound in Bud32. However, the γ-phosphate of ATP and AMPCPP are still ambiguous. Statistics for all the data collections and refinement of the different structures are summarized in supplementary Table 1.

Pcc1-Gon7 complex

The freshly purified native or SeMet-substituted Pcc1-Gon7 complex was concentrated to 25 mg/ml and applied for vapor diffusion crystallization using hanging drops. The crystals of native Pcc1-Gon7 or SeMet-labelled Pcc1-Gon7 were obtained at 18 °C from crystallization reservoir that mixed equal volume of Pcc1-Gon7 complex with solution containing 20%Glycerol, 20% PEG1500, 0.1 M Acetate Sodium pH 4.2, 3% Methanol and 0.5 M NaCl. Crystals were cyro-protected by soaking with crystallization liquor supplemented with 25% PEG400 v/v in liquid nitrogen prior to X-ray data collection. Native dataset and SAD dataset were collected at 100K on SOLEIL Synchrotron beamline Proxima 1 (Saint Aubin, France). The reflection images of the native dataset and SeMet-substituted dataset were processed at 3.0 Å and 2.5 Å, respectively, with XDS package [START_REF] Kabsch | Xds[END_REF].

Both crystals belong to space group C2 with three molecules of Pcc1-Gon7 complex in the asymmetric unit (cell parameters are summarized in Table S1).

The structure of SeMet-substituted Pcc1-Gon7 complex was determined by SAD method. 9 selenium sites (6 from Pcc1 and 3 from Gon7) out of 18 selenium sites in the total 3 molecules of Pcc1-Gon7 complex were located by using the program SHELXD [START_REF] Sheldrick | Experimental phasing with SHELXC/D/E: combining chain tracing with density modification[END_REF]. The phasing and the refinement of the Se sites were performed with Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF] and Parrot [START_REF] Cowtan | Recent developments in classical density modification[END_REF], respectively. The model building was carried out with use of Buccaneer in CCP4 package [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] and COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF], also using the structural model of P. furiosus Pcc1 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The final model of SeMet Pcc1-Gon7 complex was refined with REF-MAC5 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF]. The three copies of the complex in the asymmetric unit are identical and the model contains residues 9-90 of Pcc1 (native full length contains 1-88 plus 6xHis) and residues 3-21, 65-91 and 98-107 of Gon7 (native full length contains 1-123). The missing residues were not observed in all the crystal structures. The structure of SeMet-substituted Pcc1-Gon7 complex was used as a searching model for the native dataset using MOLREP [START_REF] Vagin | Molecular replacement with MOLREP[END_REF]) and the final model of Pcc1-Gon7 complex was built with COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]) and refined against the native dataset usiong REFNMAC5 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF]. The data collection and refinement statistics are summarized in Table 1.

Size-Exclusion-Chromatography coupled to Multi-Angle Laser Light Scattering (SEC-MALLS) measurement

The determination of the molecular weight of the proteins in solution was carried out with the SEC-MALLS (Malvern, France). The Superdex TM 75 HR 10/30 or Superdex TM 200 HR 10/30 columns (GE Healthcare) for Size-Exclusion Chromatography was equilibrated with buffer composed of 20 mM Tris pH 7.5, 200 mM NaCl and 5 mM 2-mercaptoethanol. 80-100 µl Gon7-Pcc1-his or KEOPS-His at a concentration of 3-5 mg/ml were injected at a flow rate of 0.5 ml min -1 onto the SEC column and eluted with the equilibration buffer at a flow rate of 0.3 ml•min -1 . Elution was followed by a UV-visible spectrophotometer, a differential refractometer, a 7° Low Angle Light Scattering detector, a 90° Right Angle Light Scattering detector and a differential pressure viscometer. The OmniSEC software program was used for the acquisition and analysis of the Viscotek data. BSA was used as standard reference protein of known molecular weight, concentration, refractive index increment (dn/dc = 0.185 ml•g -1 ), and intrinsic viscosity to determine the instrument response factors of the detectors for the mobile phase being used.

Small Angle X-ray Scattering (SAXS)

SAXS experiments were carried out at SOLEIL synchrotron SWING beamline (Saint-Aubin, France). The sample to detector (Aviex CCD) distance was set to 1500 mm, allowing reliable data collection over the momentum transfer range 0.008 Å -1 < q < 0.5 Å -1 with q=4πsinθ/λ where 2θ is the scattering angle and λ the wavelength of the X-rays (λ= 1.0 Å). In order to separate the isolated partners from complexes, SAXS data were collected on solutions eluting from an online size-exclusion high-performance liquid chromatography (SE-HPLCBio-SEC3 Agilent) column available on SWING and directly connected to the SAXS measuring cell. The purified KEOPS complex or Pcc1-Gon7 complex were injected in the column pre-equilibrated with preparative buffer comprising 20 mM Tris pH 7.5, 200 mM NaCl and 5 mM 2-mercaptoethanol.

Flow rate was 200 µL/min, frame duration was 1 s and the dead time between frames was 0.5 s. For each frame, the protein concentration (between 0.5 and 2 mg/mL at the top of elution peak) was estimated from UV absorption at 280 nm and 295 nm using a spectrometer located immediately upstream of the SAXS measuring cell. Selected identical frames corresponding to each elution peak were averaged. A large number of frames were collected before the void volume and averaged to account for buffer scattering. SAXS data were normalized to the intensity of the incident beam and background (i.e. the elution buffer) subtracted using the programs FoxTrot (courtesy of SWING beamline) and Primus [START_REF] Konarev | ATSAS 2.1, a program package for small-angle scattering data analysis[END_REF][START_REF] Konarev | PRIMUS: a Windows PC-based system for small-angle scattering data analysis[END_REF]. The scattered intensities were displayed on an absolute scale using the scattering by water.

In order to determine unambiguously the oligomeric state of the protein or complex the molar mass was obtained using the macromolecule volume and Gon7.The final model does not exhibit pronounced steric clashes at interfaces of each set of subunits and consistent with the mutations that disrupt the interactions [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

Results

Structure of the Gon7/Pcc1 complex

The assembly of the available structures of archaean KEOPS subcomplexes allowed reconstructing the complete complex, revealing that Pcc1, Kae1, Bud32 and Cgi121 are arranged in a linear fashion. The Kae1 subunit is situated at the center of the complex, while Pcc1 binds at its N-terminal and Bud32 at its C-terminal lobe. Cgi121 is bound to Bud32 at the opposite site of Kae1 (Fig. S1). We speculated that Gon7 would bind to one of the extremities of the KEOPS complex, forming a complex either with Pcc1 or with Cgi121.

We first co-expressed Gon7-Pcc1-His and observed that both proteins copurified and form a stable complex (Fig. S2A). We obtained good quality preparations of purified Gon7-Pcc1 that was used for the crystallization trials. The Xray crystal structure of Pcc1-Gon7 complex was determined after Semethionine labeling. There are three copies of the Gon7/Pcc1 heterodimer in the asymmetric unit. Gon7 is partially ordered since electron density could only be observed for the peptide regions comprised between residues 3 to 21, 63 to 95 and 98 to 110. The disordered regions are about the same for the three copies of Gon7 in the asymmetric unit. Gon7 is made of an anti-parallel two-stranded β-sheet (residues 3 to 21) and a long α-helix (residues 63 to 95) that are packed to form the structured core of the protein (Fig. 1A). Two copies of Gon7 in the asymmetric unit have a supplementary α-helix (residues 98 to 113) that is pointing away from the structured core, contacting the helical region of a neighboring Gon7/Pcc1 heterodimer in the crystal (Fig. 1D and1E). The structure of Pcc1 (88 residues) is well defined between residues 8

(or 9, depending on the copy) and 91 (3 histidines from the 6xHis tag). Pcc1 forms a three-stranded anti-parallel β-sheet with two helices packed on one face. The structure of yeast Pcc1 is very similar to that of P. furiosus Pcc1 [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF], with a Z-score 7.7 and an RMSD of 1.09 Å for 65 C α atoms aligned. Dimer formation between Gon7 and Pcc1 involves packing of two helices and formation of an anti-parallel β-sheet. The first β-strand of Gon7 aligns with the first β-strand of Pcc1, creating a continuous anti-parallel five stranded β-sheet. Complex formation buries about 2400 Å 2 for a total of 11700 Å 2 accessible surface area. The most important contribution to the dimer interface results from the packing of the α1-helix of Gon7 against the α2helix of Pcc1. The interface is stabilized by 15 hydrogen bonds and 5 salt bridges. The well conserved residues from the Gon7 α1 helix directly interact with Pcc1: hydrophobic residues form the core of the interface while polar and charged residues form specific hydrogen bonds at the periphery (for instance the totally conserved Arg72 of Gon7 H-bonds to Glu 83 of Pcc1). The sequence of the β1 strand of Gon7 is not well conserved but the majority of its interactions with Pcc1 consist of hydrogen bonds between main chain atoms. Archaean Pcc1 alone or in complex with Kae1 forms a tight homodimer, characterized by a continuous six-stranded anti-parallel β-sheet and packing of two helices. Pcc1 dimer formation was also observed in solution and it was suggested that Pcc1 functions as a dimerization module of the KEOPS complex in archaea (Fig. S1). Superposition of the Gon7/Pcc1 complex onto the Pcc1 homodimer reveals that their interfaces are very similar (Fig. 1B and1C). The part of Gon7 that is structured overlaps very well with the secondary structure elements of Pcc1 that are involved in homodimer formation. It should be noted that, although Gon7 and Pcc1 share the same architecture, they do not have the same fold. We conclude that Gon7 is a structural mimic of Pcc1 and precludes formation of the Pcc1 homodimer.

Gon7/Pcc1 in solution

Sequence-based struture prediction indicates that Gon7 is totally disordered (Fig. S3A). Since more than half of the Gon7 protein was not observed in the crystal, we investigated whether Gon7 becomes partially structured upon complex formation with Pcc1. We therefore prepared N 15 -labeled Gon7

and investigated the folding of the construct by NMR. The HSQC spectrum of Gon7 shows that the protein alone in solution is unstructured (Fig. S3B). We attempted to titrate labeled Gon7 with unlabeled Pcc1, but the latter protein forms stable soluble aggregates when produced alone and hence did not affect the HSQC spectrum of Gon7.

We then examined the oligomerization state of the Gon7/Pcc1 complex in solution by SEC-MALLS. The extracted molecular weight of Gon7-Pcc1-his was significantly larger than expected for a heterodimer (Fig. S3C), suggesting that Gon7/Pcc1 forms a heterotetramer in solution. We noticed the presence of two types of hetero-tetramer in the crystal packing of Gon7/Pcc1, each composed of two Gon7 and two Pcc1 subunits. In hetero-tetramer type 1 two central Gon7 proteins align their beta sheets forming a continuous 10stranded beta sheet with the Pcc1 units (Fig. 1D). The heterotetramer type 2 is created by the packing of the helical surface of two heterodimers (Fig. 1E).

The main contribution for the interaction between the heterodimers comes from the C-terminal helix of Gon7 that swaps over to the long helix of Pcc1 in the other heterodimer.

We examined the structure of the Gon7/Pcc1 complex in solution by SAXS.

The X-ray scattering curves and structural parameters are presented in Fig. S6A and Table S2, respectively. The experiments were performed using a gel filtration column coupled to the SAXS [START_REF] David | Combined sampler robot and high-performance liquid chromatography: a fully automated system for biological small-angle X-ray scattering experiments at the Synchrotron SOLEIL SWING beamline[END_REF]. SAXS data were collected online throughout the elution time. Selected identical scattering curves corresponding to the top of the elution peak were averaged.The value of the molar mass M obtained from the whole I(q) curve (M=51±5 kDa) ) is compatible with the calculated mass of the heterotetramer Gon7/Pcc1 (M calc =48.8 kDa). Unfortunately, the SAXS data do not allow choosing between the two types of heterotetramers (Fig. S6B andS6D), as both can be equally well incorporated within the envelope of the complex deduced from the SAXS curve using the program GASBOR (Fig. S6) [START_REF] Svergun | Determination of domain structure of proteins from X-ray solution scattering[END_REF].

Structure the binary complex of Bud32-Cgi12 in complex with ADP

We expressed and purified Bud32-Cgi121 complex (Fig. S2B), and determined the crystal structures for yeast complex Bud32-Cgi121 in complex with ADP (Fig. 2). Crystal structures of archaean and human Cgi121 and archaean ternary complex Kae1-Bud32-Cgi121 were previously reported [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The structure shows that Cgi121 interacts with N-terminal lobe of Bud32. The structure of yeast Cgi121 is very similar to that of its human and archaean homologs (RMSD of 0.6Å for all superposed C α atoms). Yeast

Cgi121 is made of a central four stranded β-sheet and 8 α-helices. 6 helices group into a globular bundle on one side of the β-sheet and provide the main interaction site with Bud32. Archaean Cgi121 has a shorter N-terminal region compared to Cgi121 from human and yeast and therefore its central β-sheet only contains three strands compared to four in the eukaryotic structures. The N-terminal lobe of Bud32 interacts with Cgi121 and the C-terminal lobe interacts with Kae1 as shown in crystal structures of Kae1/Bud32 and Kae1/Bud32/Cgi121 archaean complexes [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The fourth β-strand from the N-terminal lobe of the Bud32 fits into the space created by the parallel α2-and α6-helices from Cgi121. The structure of the binary yeast Bud32/Cgi121 complex is similar to that described in the context of the archaean Kae1/Bud32/Cgi121 ternary complex [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF] but with a few pronounced differences (Fig. S4). First, additional contacts are made between Bud32 and Cgi121 in the yeast complex involving residues 4 to 10 from Cgi121 (connecting strands 1 and 2) and residues 35 to 42 from Bud32. The latter connection has an insertion of 11 residues compared to archaean homologs. The contact surface of the yeast Bud32-Cgi121 complex (about 1175 Å 2 ) is therefore larger than for its archaean counterpart (700 Å 2 ).

The Cgi121-Bud32 interface is composed of both hydrophobic and polar interactions (9 hydrogen-bonds). A second important structural difference with the archaean complexes resides in Bud32. Bud32 is a small atypical protein kinase with a shortened C-lobe lacking the activation segment. The structure of yeast Bud32 is well defined in the complex, except for the disordered connection (residues 58 to 68) between the third β-strand and the second α-helix.

We were unable to obtain crystals for yeast Bud32 alone and hence ignore whether conformational changes are induced by Cgi121 binding. We compared the crystal structures of Bud32 as present in yeast Bud32/Cgi121 with those from the archaean binary and ternary complexes. Generally, the Nterminal lobe of protein kinases is composed of 5 strands and a flanking αhelix. The first strand of Bud32 was found to be disordered in all reported structures of archaean Bud32 but is well defined in the yeast Bud32/Cgi121 complex. The N-terminus of Bud32 forms a short α-helix followed by a loop that contacts Cgi121 and that is absent in its archaean homologs (Fig. S4).

Although both the N-and C-terminal lobes of yeast and archaeal Bud32 are superposed well individually, their relative orientations are different. Upon superposition of the N-terminal lobes of yeast and archaean Bud32, their Cterminal lobes are slightly shifted (data not shown).

The structure of the M. jannaschii Kae1/Bud32 complex was determined in the presence of the nucleotide analogue AMPPNP. Although the electron density for AMPPNP was clearly defined in the active site of Kae1, it was absent from Bud32 [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF]. Likewise, no binding of nucleotide was observed in Bud32 in the structure of the ternary Kae1-Bud32-Cgi121 complex from archaea [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. We cocrystallized yeast Bud32/Cgi121 in presence of ATP, ADP, AMPPNP and AMPCPP and in all cases the nucleotide was well bound at the active site of Bud32 (Fig. 2). The adenosine base occupies a hydrophobic pocket created by residues of the linker region between the two lobes. For all nucleotides the α-and β-phosphates are well defined and in some cases bound to a divalent Mg ion, which is tightly coordinated by Asp 161 . The α-and β-phosphate groups further interact with Lys 52 , a very conserved residue in the active site of kinases that is involved in the stabilization of the penta-covalent transition state during the phosphorylation reaction. The metal ion is further liganded to the side chains of Asn 166 and Asp 182 and close to the conserved Asp 180 (Fig. 2 and Fig. S4 and Fig. S5).

The P-loop between β1 and β2 strands of Bud32 (Ser 23 -Ile 29 ) was disordered in structures of archaean Bud32 in the absence of nucleotides but becomes ordered in yeast Bud32 when the nucleotides are bound. We did not observe electron density for the γ-phosphate for any of the nucleotide complexes. We presume the γ-phosphate is labile since it was neither observed in the nonhydrolysable AMPPNP analog. The presence of the nucleotide in the active site of the yeast Bud32 structure is coherent with the ordered β1-strand and P-loop. The mutations of catalytic cleft residues that destroy the autophosphorylation activity of Bud32 and cause cell growth and telomere length defects (Lys 52 , Asp 182 , Asp 161 ) are all directly bound to the ADP in Bud32 [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF][START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Also, Ser 187 and Ser 189 that are autophosphorylation sites of Bud32 reside on the loop which is accessible to the ADP binding site (Fig. 2 and Fig. S4) [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF].

Model of the complete yeast KEOPS complex

A model of archaean KEOPS was previously proposed based on the structures of subcomplexes [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. We used this approach to construct a model of yeast KEOPS by superposing the present Bud32-Cgi121 and Pcc1-Gon7 structures onto their orthologs in archean KEOPS. We incorporated a yeast Kae1 model that was obtained by Phyres 2 [START_REF] Kelley | Protein structure prediction on the Web: a case study using the Phyre server[END_REF].. The model for the complete yeast KEOPS complex is represented in The crystal structures of archaean Pcc1 alone and of the Pcc1/Kae1 complex showed that Pcc1 forms homodimers [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. It was therefore suggested that the intact archaean complex would form a dimer of heterotetramers for which Pcc1 would function as the dimerization unit [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. The structure of the Gon7/Pcc1 dimer shows that Pcc1 occupies the same interface as that used for Pcc1 homodimer formation (Fig. 1B and1C).

We therefore set out to determine the quaternary structure of the complete yeast KEOPS complex. Recombinant yeast KEOPS complex was puritied after high yield expression from a polycistronic in E. coli cells (Fig. S1C). We first determined the molecular mass in solution of the recombinant yeast KEOPS complex by SEC-MALLS. The extracted molecular weight was 109.7 kDa (Fig. 4A), which is close to the theoretical molecular weight of the pentameric KEOPS complex (117.7 kDa), suggesting yeast KEOPS forms a hetero-pentamer in solution. In order to confirm the stoichiometry and to evaluate our model of the complex, we investigated the intact KEOPS complex in solution by SAXS coupled to gel filtration. The molar mass extracted from the scattering curve corresponding to the top of the elution peak is equal to 122±10 kD (Table S1), in perfect agreement with the MALLS results. We evaluated our model of the yeast KEOPS complex against the SAXS data.

The program BUNCH [START_REF] Petoukhov | Global rigid body modeling of macromolecular complexes against small-angle scattering data[END_REF] was used for modeling the missing parts of the model (mainly in the Gon7 subunit) as chains of dummy residues that are separated by 3.8 Å. Adding the side chains using the program PD2 [START_REF] Moore | High-quality protein backbone reconstruction from alpha carbons using Gaussian mixture models[END_REF] then completed the models. The scattering amplitude curves calculated on the final model by using the program CRYSOL [START_REF] Svergun | CRYSOL -a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates[END_REF] is in excellent agreement with the experimental curve (χ=1.25) (Fig. 4B). These data validate the quaternary structure and the molecular architecture of our model of yeast KEOPS.

Discussion

Experimental data obtained from archaean and eukaryotic t 6 A biosynthesis systems led to the conclusion that the KEOPS complex catalyzes the transfer of the threonylcarbamoyl moiety from the unstable TCA intermediate onto the A 37 of acceptor tRNA [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The active sites of the t 6 A biosynthesis systems are situated on the very conserved metal cluster in Kae1/Qri7/YgjD proteins. The role of the other subunits (Bud32/Pcc1/Cgi121/Gon7) remains, however, largely unknown.

Analysis of the archaean KEOPS complex showed that Pcc1, Bud32 and Kae1 are the minimal protein set for the in vitro t 6 A biosynthesis but that Cgi121 increases the efficiency of the reaction [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Bud32

was shown to possess both ATPase and kinase activities [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Active site mutations of Bud32 (D161A/R in yeast and D127R in archaea) affect kinase activity [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF][START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF], t 6 A biosynthesis activity [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF] and ATPase activity [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. The threonylcarbamoyl-transfer from TCA onto A 37 of tRNA does not in principle require ATP hydrolysis nor the kinase activity of Bud32. This was clearly demonstrated by the study of the mitochondrial Qri7 enzyme which is active without assistance of other proteins, forming together with Sua5 the minimal t 6 A biosynthesis system.

Though AMPPNP was present in the crystallization, no nucleotide was bound in Bud32 in the structures archaean Bud32 [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF], which was explained by its disordered N-terminus and P-loop region.

ATP is firmly bound in the yeast Bud32-Cgi121 complex interacting in a canonical manner with the well-structured P-loop. Kae1 and Cgi121 seem to have antagonistic effects on the ATPase activity of Bud32. Bud32 in complex with Cgi121 was also shown to have autphosphorylation activity [START_REF] Miyoshi | Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein[END_REF], which was proven to be inhibited by increasing concentrations of Kae1 [START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF].

Bud32 belongs to the RIO-type family of atypical small kinases that lack the ATPase during the t 6 A synthesis reaction. We hypothesize that Bud32 might regulate the interaction of the KEOPS complex with substrate tRNA. The positioning of the Bud32 subunit within our model of the KEOPS complex supports this hypothesis. The C-terminus of Bud32 lies at the entrance of the active site of Kae1. It is composed of a very positively charged helix whose sequence is extremely well conserved in eukaryotes (Fig. 3B and Fig. S5). We therefore expect that Bud32 might interfere with the binding of the bulky tRNA substrate in the Kae1 active site. ATPases often undergo conformational transitions be-tween the ATP and ADP bound states. ATP hydrolysis could influence the relative position of the N-terminal and C-terminal lobes of Bud32 and hence change for instance the position of the C-terminal helix, which in turn may modify its interactions with the substrate tRNA during t 6 A biosynthesis.

Mao et al. used the structure of archaean Kae1/Bud32/Cgi121 complex to study the functional effects of complex disrupting mutations in yeast [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Interface mutations (L172E/A176R) that interrupt interaction between Cgi121 and Bud32 displayed slow growth phenotype, analogous to the CGI121 gene deletion mutant. The present structure of the yeast Bud32/Cgi121 complex confirms that the interfacial residues targeted in Mao's study make crucial interactions in the yeast complex and hence these functional data are in full agreement with our structure and model.

Gon7, the fifth fungus-specific KEOPS subunit, was predicted to be largely unstructured (Fig. S3A), which we confirmed by NMR measurements (Fig. S3B). We showed here that it forms a tight complex with Pcc1, which functions as a dimerization unit of the archaeal KEOPS complex. Also the human KEOPS complex was reported to form a dimer of heterotetramers [START_REF] Costessi | The Human EKC/KEOPS Complex Is Recruited to Cullin2 Ubiquitin Ligases by the Human Tumour Antigen PRAME[END_REF]. Upon complex formation with Pcc1, about half of Gon7 acquires a well-defined structure and forms a continuous β-sheet structure with Pcc1.

The binding of Gon7 to Pcc1 is incompatible with Pcc1 homodimer formation.

Pcc1 can therefore not function as a dimerization unit in the yeast KEOPS complex, which we experimentally confirmed by SAXS and MALLS data (Fig. 4). Combining the quaternary structure information of yeast KEOPS, the Pcc1-Gon7 heterotetramer type 2 (Fig. 1E and Fig. S6D) is possibly a dimerization mode since the type 1 could mediate the formation of a dimer of KEOPS pentamer, which was clearly not the case (Fig. S6B andC).

Our model of the complete yeast KEOPS which is compatible with our SAXS data (Fig. 3 and Fig. 4) has all subunits arranged in a linear fashion with Gon7 on one end and Cgi121 at the other. This model suggested that the highly conserved positively charged C-terminus of Bud32 might be involved in tRNA binding since it is bound very closely to the active site entrance of Kae1, near the metal binding center (Fig. 3A and3C). The region of the KEOPS complex around the ATP binding site of Kea1 is very positively charged (Fig. 3B), favorable for tRNA binding. The presence of the C-terminal helix of Bud32 near the active site of Kae1 offers an opportunity to couple ATP hydrolysis by Bud32 and tRNA binding by the KEOPS complex. The conversion of ATP into ADP in the active site might change the conformation of Bud32

and affect the position of the C-terminal helix for instance. Many RNA-binding processes are regulated by nucleotide triphophosphate hydrolysis. For instance, the elongation factor EF-Tu undergoes a conformational change upon GTP hydrolysis that dissociates its complex with aminacyl-tRNA during peptide hydrolysis on the ribosome [START_REF] Nilsson | Elongation factors on the ribosome[END_REF].

Conclusion

In the present study, we reconstituted a structural model of yeast KEOPS protein complex by using crystal structures of subcomplexes of Gon7/Pcc1

and Bud32/Cgi121. The comparison of the structural models between yeast KEOPS and its archaean counterpart reveals that Gon7 structurally mimics Pcc1 upon binding to Pcc1 and therefore prevents the yeast KEOPS from dimerizing at the Pcc1 homodimer interface. The analysis on the quaternary structure of the yeast KEOPS by SAXS confirms that yeast KEOPS exists as heteropentamer with a molecular arrangement presented by the model. In addition, the crystal structure of binary complex Bud32/Cgi121 in complex with ADP demonstrates that ADP is bound in a manner characteristic of that in PKA kinases and further provides a basis for the kinase and ATPase activities Bud32.This new yeast KEOPS structural model displays a conserved and positively charged surface formed by Bud32, Kae1 and Pcc1, providing insights into the binding of tRNA and t 6 A biosynthesis. Further experimental investigation on the assembly of the KEOPS and tRNA binding is needed to unravel its role in the biosynthesis of tRNA t 6 A, telomere elongation and transcriptional regulation.

Fig. S3 The quaternary structure of Gon7 and the Pcc1-Gon7 complex in solution. A. Sequence-base prediction on protein foldedness by FoldIndex© [START_REF] Prilusky | FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded[END_REF] indicates that Gon7 is totally unfolded. B. The NMR HSQC spectrum of Gon7 demonstrates that Gon7 is unstructured. C. The gel filtration profile and molecular weight of Pcc1-Gon7 complex was determined to be 34.6 kD by SEC-Malls, larger than the a calculated molecular weight of 24.4 kD.

Fig. S4

The structural comparison of S. cerevisiae Bud32 with M. jannaschii Bud32. S. cerevisae Bud32 (in blue) is structurally superposed to M. jannaschii Bud32 from MJ1130 (in green) and ternary complex Kae1/Bud32/Cgi121 (in orange). The mutation sites that are discussed in the text are labeled.

Fig. S5

The sequence alignment of Bud32 from S. cerevisiae (gi|6321701|NP_011778.1), P. abyssi (gi|14521787|NP_127263.1), A. thaliana (gi|18420990|NP_568482.1), M. musculus (gi|31560052|NP_076304.2), H. sapiens (gi|41327715|NP_291028.3) and M. jannaschii (gi|15669317|NP_248122.1). Secondary structure of S. cerevisiae Bud32 is shown at the top. The sequence alignments were obtained using Clustal Omega [START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF] and ES-Pript [START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF]. The red stars denote the positions of the muations highlighted in Fig. S4.

Introduction

During maturation tRNAs undergo many post-transcriptional modifications, some of which are essential for cell life [START_REF] Phizicky | Do all modifications benefit all tRNAs?[END_REF][START_REF] Yarian | Accurate translation of the genetic code depends on tRNA modified nucleosides[END_REF]. The N 6 -threonylcarbamoyladenosine (t 6 A) modification at position 37 of ANN-decoding tRNAs is one of the few modifications that are found in the three domains of life [START_REF] Chheda | Isolation and characterization of a novel nucleoside, N-[(9beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]threonine, from human urine[END_REF][START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF][START_REF] Grosjean | Deciphering synonymous codons in the three domains of life: co-evolution with specific tRNA modification enzymes[END_REF]. The t 6 A base on E. coli tRNA Lys stacks with its adjacent A 38 and forms a cross-strand stack with the first codon of the mRNA, contributing to the translational fidelity [START_REF] Durant | Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A[END_REF][START_REF] Fvt | The role of modifications in codon discrimination by tRNA(Lys)UUU[END_REF]. Furthermore, several isopentenyl-adenosine derivatives at position 37, such as N 6 -methyl-N 6 -threonylcarbamoyladenosine (m 6 t 6 A) and cyclic t 6 A (ct 6 A), are derivatives of t 6 A in some tRNAs [START_REF] Miyauchi | A cyclic form of N(6)threonylcarbamoyladenosine as a widely distributed tRNA hypermodification[END_REF][START_REF] Qian | The methyl group of the N6-methyl-N6threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA[END_REF]. Absence or defective t 6 A has been extensively implicated in compromised anticondon-codon interaction, erroneous selection of start codons and aberrant frameshift as well as numerous pleiotropic phenotypes [START_REF] El Yacoubi | Biosynthesis and function of posttranscriptional modifications of transfer RNAs[END_REF][START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF]. It has been shown that the biosynthesis of t 6 A proceeds in two main steps: in the first, members of the Sua5/YrdC protein family, present in bacteria, eukaryotes and archaea, utilize L-threonine, bicarbonate and ATP to synthesize an unstable intermediate threonlycarbamoyladenylate (TCA) [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF] In the second step the threonlycarbamoylmoiety of TCA is transferred onto A 37 of substrate tRNA [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. In bacteria, the transfer involves three proteins: YgjD, YeaZ and YjeE from E. coli (B. subtilis orthologs YdiE, YdiC and YdiB) [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF]. In archaea and yeast, the transfer reaction requires the KEOPS protein complex, composed of Kae1, Bud32, Cgi121, and Pcc1, complemented by a fifth fungi-specific protein Gon7 in yeast [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]Kisseleva-Romanova et al, 2006b;[START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF] -. It is now well established that YgjD and its ortholog Kae1 are responsible for the catalysis of the transfer reaction. The contribution of the other protein partners is indispensable but their biochemical function is poorly understood [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. For example, the mutation of catalytic residues of Bud32, an atypical P-loop kinase [START_REF] Facchin | Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB[END_REF][START_REF] Lopreiato | Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin[END_REF], abolished the formation of tRNA t 6 A in vitro and resulted in severe cell growth [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF], but the need of a kinase activity remains for the moment unexplained.

Interestingly, the yeast mitochondrial YgjD/Kae1 paralog Qri7 is capable of in vitro biosynthesis of tRNA t 6 A on its own if provided with TCA [START_REF] Oberto | Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The active site of YgjD/Kae1/Qri7 is characterized by a conserved metal-cluster that is contacting the phosphate moieties of a bound ATP (Hecker et al, 2007b;[START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF][START_REF] Tominaga | Structure of Saccharomyces cerevisiae mitochondrial Qri7 in complex with AMP[END_REF] .

Mutations of this metal-binding motif abolish the biosynthesis of t 6 A [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Mutations or deletions of KEOPS subunits that affect the biosynthesis of tRNA t 6 A have wide ranging biological effects in yeast such as telomere stability, transcription and genomic stability (Daugeron et al, 2011b;[START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]Kisseleva-Romanova et al, 2006b;[START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. It has not been established yet whether KEOPS is directly involved in these processes or whether the phenotypical effects of the deletion mutants are due to impaired t 6 A synthesis.

The essential ygjD, yeaZ and yjeE genes are present in all bacterial genomes, exception made for a few organisms with highly reduced genomes such as C. ruddii and S. muelleri [START_REF] Arigoni | A genome-based approach for the identification of essential bacterial genes[END_REF][START_REF] Freiberg | Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria[END_REF][START_REF] Mccutcheon | Extreme genome reduction in symbiotic bacteria[END_REF]. The YeaZ (231 residues) protein is a truncated paralog of YgjD (337 residues), having no close orthologs in eukaryotes. A direct interaction between YgjD and YeaZ from E. coli has been documented

and the crystal structure of heterodimer YgjD-YeaZ from S. typhimurium was determined [START_REF] Butland | Interaction network containing conserved and essential protein complexes in Escherichia coli[END_REF][START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. The structural comparison of the YgjD-YeaZ heterodimer and the YeaZ homodimer revealed that the same regions of YgjD and YeaZ are involved in dimer formation [START_REF] Nichols | Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog[END_REF][START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. Furthermore, the interacting interface of YgjD-YeaZ is structurally similar to those of the Kae1-Pcc1 complex and of the Qri7 homodimer [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Bacterial two hybrid studies suggested that YeaZ is capable of interacting with either YjeE or YgjD, forming an essential network that is regulated by ATP turnover by YjeE [START_REF] Handford | Conserved network of proteins essential for bacterial viability[END_REF][START_REF] Msadek | Grasping at shadows: revealing the elusive nature of essential genes[END_REF]. YjeE is only present in bacteria and was reported to possess an intrinsically weak ATPase activity [START_REF] Allali-Hassani | Probing the active site of YjeE: a vital Escherichia coli protein of unknown function[END_REF][START_REF] Freiberg | Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria[END_REF][START_REF] Handford | Conserved network of proteins essential for bacterial viability[END_REF]. The crystal structure of YjeE from H. influenzae in complex with ADP reveals the presence of a Walker A motif and two switch regions that are characteristic of P-loop GTPases [START_REF] Brown | Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology[END_REF][START_REF] Teplyakov | Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis[END_REF]. The hydrolysis of ATP into ADP is observed only when YgjD, YeaZ and YjeE are mixed, regardless of the presence of other substrates for biosynthesis of tRNA t 6 A [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF]. The contribution of the hydrolysis of ATP to the overall biosynthesis of t 6 A in bacteria is presently unclear.

We report here the crystal structure of the heterodimer YgjD-YeaZ from E.

coli in complex with ADP. We characterized the activation and regulation of the ATPase activity of YjeE by the heterodimer YgjD-YeaZ . The nature of this atypical ADP-binding site at the YgjD-YeaZ interface suggests this might also be the binding site for YjeE. We made a 3D model of the YgjD-YeaZ-YjeE complex that is compatible with experimental SAXS data. Furthermore, we

show that the Mg 2+ -binding site in the putative catalytic center of YgjD is essential for biosynthesis of tRNA t 6 A. Eventually, we demonstrate that the ATPmediated dynamic formation of the ternary complex YgjD-YeaZ-YjeE is required for tRNA t 6 A modification but not its ATPase activity.

Experimental

Cloning, mutagenesis and expression E. coli genes ygjD, yeaZ, yjeE, and yrdC were amplified from E. coli K-12 genomic DNA and cloned into vectors pET9a, pET9a, pET28a and pET26b, respectively [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Shortly, ygjD, yeaZ and yrdC were subcloned into the corresponding vectors above with an addition of 6xHis tags at the C-terminus whereas yjeE was subcloned into its vector with an addition of

Strep II tag at the C-terminus. YgjD, YeaZ, YjeE and YrdC were produced at 37 °C in E. coli BL21(DE3) Competent Cells (Invitrogen) transformed with pET9a-ygjD, pET9a-yeaZ and, pET28a-yjeE and pET26b-yrdC, respectively.

The yields are around 1 mg per liter for all the four proteins.

The following mutations have been inserted using the Quikchange mutagenesis kit (Agilent Technologies): YgjD-F100E; YgjD-S97E; YgjD-S97R;

YgjD-E12A; YgjD-V85E; YeaZ-Δ220; YeaZ-R118A; YjeE-E108A; YjeE-T43A;

YjeE-Y82A and YjeE-W109A. Primers (sequences are summarized in supporting materials) have been ordered from Eurofins Genomics, Les Ulis, France. Plasmid template (10 ng) has been incubated with 0.04 µM of each forward and reverse primer, 1 µM of dNTP mix (Thermo Scientific), 1 unit of Phusion High Fidelity DNA polymerase (Thermo Scientific) in HF buffer. 25 cycles of amplification have been carried out, corresponding to 1 min of denaturation at 95°C, 1 min of annealing at 55°C and 2'30'' of extension at 72°C.

PCR tubes have been incubated for 5 more min at 72°C as a final extension step. Parental plasmid has been digested for 2 hours at 37°C with DpnI restriction enzyme (Thermo Scientific) and 5 µL of the PCR mix have been used

to transform XL10 chimio-competent cells (Agilent Technologies). For the screening procedure, 4 to 8 independent colonies have been grown in LB medium supplemented with the appropriate antibiotic and plasmid have been extracted using the Genejet plasmid miniprep kit (Thermo Scientific). Plasmids have been sequenced (Beckman Coulter Genomics) on the whole gene to check for the presence of the right mutation. In few cases an alternative protocol has been used where two separate PCRs have been carried out in parallel using only the forward and the reverse primer in two separate tubes and 100 ng of plasmid template. In this case 30 cycles of amplification have been done and the two tubes have been pooled, heated to 95°C and slowly cooled down before DpnI digestion [START_REF] Edelheit | Simple and efficient sitedirected mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies[END_REF]. The rest of the protocol was the same as the one mentioned above. In all cases, all the genes have been sequenced on their full length.

The expression and purification details of all proteins are presented in the supporting materials (SM) sections.

Native gel migration assay

A 1.2% agarose gel was prepared in 30 mM Tris-HCl pH 8.0 and 50 mM Glycine. Protein samples supplemented with 25% glycerol were loaded onto the gel. Migration lasted for 50 min at 135 mV in a running buffer composed of 30 mM Tris-HCl pH 8.0 and 50 mM Glycine. For analysis of the ATP/ADPdependent association of the proteins, both the gel and the loading protein samples were additionally supplemented with 1.0 mM of ATP or ADP and 2.0 mM MgCl 2 . The gel was running in an electrophoresis apparatus on ice with an ambient temperature at 8 °C. Proteins were revealed by Coomassie staining.

Biophysical and structural analysis

Crystallography, SAXS and biophysical measurements (ITC) are presented in the supporting materials section.

ATPase activity assay

The ATPase activity (conversion into ADP and phosphate) was measured using a NADH-coupled assay [START_REF] Tamura | Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine[END_REF]. The hydrolysis of ATP into ADP was coupled to the oxidation of NADH to NAD + and the consumption of NADH was collected by monitoring the absorbance at A 340 by a Cary WinUV 200 spectrophotometer (Agilent Technology, Inc.). Assays were performed in a 10mm cuvette at 20 °C. The 150 µL reaction buffer contained 50 mM Hepes pH 7.5, and an excess of pyruvate kinase and lactate dehydrogenase (Sigma). The reactions were monitored after the steady-state reaction was achieved and the linear curves of absorbance at A 340 were recorded at for 30 min. The concentration of the proteins in the assays was 1 µM except for YjeE and its variants, which were at 2 µM. The reaction velocity was calculated by converting the decrease in the absorbance to consumption of NADH using an extinction coefficient of ɛ 340nm =6220 M -1 •cm -1 . ATPase reaction rates as a function of ATP concentration was fitted using Michaelis-Menten equation and the cooperactivity of ATPase activity of YjeE by the dimer YgjD-YeaZ was fitted by Allosteric sigmoidal kinetics equation using the Prism program (GraphPad). All the experiments were done in triplicate and control experiments were carried out in which either proteins or chemicals were omitted from the reaction.

tRNA overexpression and purification

The E. coli tRNA Lys(UUU) was subcloned in a pBlue-Script vector harboring T5 promoter/lac operator sequence [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. The pB-tRNA Lys(UUU) was transformed into E.coli XL10 competent cells and was grown in 10ml 2xYT medium at 37C overnight. The 10 ml preculture was transferred to 800 ml Overnight Express Instant TB medium (Novagen) and the overexpression was auto-induced during the culturing for 36 hrs at 37°C. The cell culture was harvested and centrifuged for 25 min at 4400 rpm. The cell pellets were suspended in Trizol (Sigma), followed by extraction of the total RNAs according to the manufacturer's procedure (Tri Reagent, Sigma). The total RNA pellet was dissolved in water and applied for purification by a denaturing gel that is composed of 10% of polyacrylamide and 8M Urea. After migration for 4 hours at 250V, 25mA in TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA), the gel slice containing tRNAs as visualized by UV light shadowing at 258nm and was cut out and incubated with elution buffer (10 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA and 1% SDS) overnight at an ambient temperature. The eluted tRNAs were precipitated with absolute ethanol and NaCl. Finally, the tRNAs were further purified by size exclusion chromatography equilibrated with buffer that is composed of 10 mM Hepes pH 7.5, 100 mM NaCl. The size and purity of the tRNA was analyzed by 1.5% agarose gel.

The tRNA t 6 A assay

The in vitro biosynthesis of tRNA t 6 A, the digestion of tRNA and the HPLC/MS analysis of the t 6 A formation used published protocols and are presented in the supporting materials (SM) section.

Results

YgjD forms a stable heterodimer with YeaZ

The four E. coli proteins YrdC, YgjD, YeaZ and YjeE are necessary and sufficient for the in vitro synthesis of tRNA t 6 A [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF]. The coexistence of homodimers and monomers of YgjD, YeaZ and YjeE have been observed and YgjD is capable of interacting with YeaZ, during which activation of the proteolysis of YgjD occurs [START_REF] Handford | Conserved network of proteins essential for bacterial viability[END_REF]. In order to get insight into the biochemical function of YrdC, YgjD, YeaZ and YjeE we decided to investigate their interactions in vitro. We first expressed and purified the four recombinant proteins separately. The C-terminal 6xHis-tagged YgjD is stable and is not sensitive to proteolysis as reported previously [START_REF] Handford | Conserved network of proteins essential for bacterial viability[END_REF]. We tested the capability of various combinations of proteins to form complexes by native gel shift experiments. A mobility shift is observed upon mixing YgjD and YeaZ, demonstrating they are spontaneously forming a complex (Fig. 1A, lanes 1-4). No other combination of proteins provoked shifts in the migration.

Fig. 1 The in vitro characterization of protein-protein interactions between YgjD, YeaZ, YjeE and YrdC. A. The native agarose gel shift analysis of various protein mixtures. + signs define the composition of the protein mixtures. The molar ratio in each mixture was 1:1, except * , which was ½ for YgjD. B. Superposed size exclusion chromatographic profiles of YgjD, YeaZ, YgjD-YeaZ and YgjD S97E .

In order to further characterize the complex formed by YgjD and YeaZ, we submitted it to size exclusion chromatography. These data confirm that YgjD and YeaZ alone form homodimers and a heterodimer is readily formed upon equimolar mixing of YgjD and YeaZ (Fig. 1B). The interaction between YgjD and YeaZ was quantified by ITC (Table 1 and Fig. S1). Despite the fact that both YgjD and YeaZ form homodimers the titration of YgjD with YeaZ in absence of nucleotides (ADP or ATP) resulted in a binding curve that could be fitted by a simple equilibrium scheme. (YgjD-YeaZ complex with 1:1 stoichiometry and a Kd of 122 nM). These binding parameters are similar as for the interaction between YgjD and YeaZ from S. typhimurium (Kd of 300 nM) [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF].

Table 1 The thermodynamic parameters for the protein-protein interaction measured by ITC.

The corresponding thermograms are presented in Fig. S1 for the upper panel and Fig. S5 for the lower panel. a , in presence of 1.0 mM AMPPNP and MgCl 2 . Crystal structure of the heterodimer YgjD-YeaZ in complex of ADP

Titrant

We reconstituted the YgjD-YeaZ complex by mixing them in a 1:1 stoichiometric ratio, followed by a size-exclusion chromatography step. We only obtained good crystals of the YgjD-YeaZ heterodimer in presence of ATP or ADP and MgCl 2 . The crystals diffracted up to 2.3 Å at the Proxima 1 beamline of the SOLEIL synchrotron. Diffraction data were indexed and processed in space group P1 (crystallographic data are gathered in Table S1). The structure of the complex was solved by molecular replacement using the structures of Pa-Kae1 and Ec-YeaZ as search models and was refined to R work/free values of 18%/23%. Crystal structures of the heterodimer YgjD-YeaZ in complex with ATP, AMP were also determined (Table S1).

YgjD and YeaZ both belong to the ASKHA (acetate and sugar kinase/HSP70/actin) superfamily (Fig. 2A) [START_REF] Hurley | The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism[END_REF]. As for Kae1 and Qri7, the conserved active site of YgjD is situated at the interface of two similar α/β-domains (Hecker et al, 2007b;[START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. The overall structure of the YgjD-YeaZ heterodimer is very similar to that of YgjD-YeaZ from S. typhimurium, with an rmsd of 0.232 Å for 571 superposed C α atoms [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. The binding modes of nucleotides (ATP, ADP and AMP) are conserved in Pa-Kae1, St-YgjD and Ec-YgjD. The interface between YgjD and YeaZ resembles that of the most commonly observed YeaZ homodimer (Fig. 2A) [START_REF] Aydin | Structural analysis of the essential resuscitation promoting factor YeaZ suggests a mechanism of nucleotide regulation through dimer reorganization[END_REF][START_REF] Nichols | Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog[END_REF]. The main part of the interface is made of a four helical bundle created by the packing of α2 and α3 from YgjD and α1 and α2 from YeaZ. The complex buries 2832 Å 2 , which amounts to about 10% of the available surface area. The interface contains well-conserved hydrophobic residues and is stabilized by 9 hydrogen and 2 salt bridge bonds (between Asp38 on the α1 of YgjD and Lys226 on the Cterminus of YeaZ). α1 of YeaZ further interacts with the C-terminal tail of YgjD, which was reported to be critical for the interaction with YeaZ [START_REF] Lei | The Cterminal domain of the novel essential protein Gcp is critical for interaction with another essential protein YeaZ of Staphylococcus aureus[END_REF]. The structures of YeaZ present in the YeaZ homodimer or the YgjD-YeaZ heterodimer are identical, except for the C-terminus of YeaZ (residues 220-231) that is disordered in the homodimer but well-structured through interaction with YgjD in the heterodimer, capping over its α1 and α2 helices.

The YgjD-YeaZ interface closely resembles that of the Qri7 homodimer (Fig. 2G). Interestingly the Kae1 interface region of the Kae1-Pcc1 complex is the same as for YgjD-YeaZ (Fig. 2H). Pcc1 is not related to YeaZ, but its binding to Kae1 strongly mimics that of YgjD and YeaZ. This suggests that, comparable to the essentiality of Qri homodimerization, YeaZ and Pcc1 could play a similar role through their interaction with YgjD and Kae1, respectively.

The YgjD/YeaZ interface creates an atypical ADP-binding site

YgjD-YeaZ cocrystallized with ATP showed clear electron density for a nucleotide bound to two metal ions in the active site of YgjD, which we interpreted as Mg and Fe based on electron density levels and X-ray fluorescence scanning at the Fe-edge (data not shown). Fe is liganded by the conserved His111, His115 and Asp300 residues. The β-phosphate group of the nucleotide completes the Fe coordination sphere (Fig. 2D and Fig. S2B). The Mg ion is bound at 6.2 Å from the Fe ion and is liganded by the carboxylate side chains of Asp11 and Glu12. It is also close (3.7 Å) to the β-phosphate oxygen of the nucleotide. Two more water molecules are involved in the coordination of the Mg 2+ ion (Fig. 2D and Fig. S2B). We did not observe electron density for the γ-phosphate group of ATP, which probably has been converted into ADP during the crystallization process. The ADP bound in YgjD is well superposable to the ATP bound in Pa-Kae1. The Fe 3+ ion in Pa-Kae1 is coordinated by a tyrosinate bond. This tyrosine is conserved in eukaryotic and archaean Kae1 orthologs but not in bacteria, nor in the Qri7 mitochondrial ortholog. The Fo-Fc difference map calculated after structural refinement revealed an important residual electron density cloud situated at the YgjD-YeaZ interface (Fig. 2C). An ADP molecule could unequivocally fit the density and its refined B-factors were similar to those of the surrounding side chains. ADP is bound on top of the YeaZ helical bundle at the heterodimer interface. The ADP nucleotide interacts mainly with the N-terminal domain of YeaZ, but there are also a few interactions with YgjD. The α-phosphate moiety of this ADP is very close to the best conserved sequence motif 64 GPGS(Y/F)TG(I/L/V)R 72 of YeaZ, reminiscent of a phosphate-binding motif [START_REF] Xu | Structure of an essential bacterial protein YeaZ (TM0874) from Thermotoga maritima at 2.5 A resolution[END_REF]. The βphosphate is forming an ionic interaction with YeaZ Arg118 and the ribose 2'and 3'-OH groups are forming hydrogen bonds with the YeaZ His34 side chain and the main chain carbonyl of YeaZ Arg32 , both these residues are also very well conserved. The adenine base lies on top of a hydrophobic depression created at the interacting interface of YgjD-YeaZ (YeaZ Thr69 , YgjD Leu89 ) but does not form any specific H-bond interactions with the heterodimer. Upon superposing the YgjD-YeaZ heterodimer and the YeaZ homodimer, we noticed that there is a large conformational change in the YeaZ loop connecting α3 and α1 (Fig. 2B residues Cys 30 -Thr 35 ). While this loop is in an open conformation in the heterodimer YgjD-YeaZ, it adopts a closed conformation in the YeaZ homodimer that overlaps with the interfacial ADP binding site in the heterodimer YgjD-YeaZ [START_REF] Nichols | Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog[END_REF][START_REF] Stelter | A complement to the modern crystallographer's toolbox: caged gadolinium complexes with versatile binding modes[END_REF] Nucleotide binding to the YgjD, YeaZ, the heterodimer YgjD-YeaZ and

YjeE

In order to better understand the role of ATP in the transfer of the threonylcarbamoyl-moiety from TCA onto tRNA, we characterized the nucleotidebinding properties of the YgjD, YeaZ, YjeE and YgjD-YeaZ by ITC. Thermo-grams (Fig. S3) and thermodynamic values (Table 2) were all obtained in presence of 2mM Mg 2+ . YeaZ did not bind to any of the nucleotides tested.

AMPCPP binds to YgjD with a Kd of 1.6 µM and a ΔH of 2.45 Kcal/mole, and to the YgjD-YeaZ heterodimer with a Kd of 0.7 µM and a ΔH of 0.9 Kcal/mole.

The heterodimerization with YeaZ does not seem to significantly affect the affinity of YgjD for ATP. We did not detect any heat exchange signal for titration of YgjD, YeaZ or YgjD-YeaZ with ADP (Fig. S3A-C). Hence, the binding of ADP to the YgjD-YeaZ interface could not be detected by ITC experiments. YjeE is structurally related to small GTPases (Fig. S2C andS2E) and exhibits intrinsically weak ATPase activity [START_REF] Allali-Hassani | Probing the active site of YjeE: a vital Escherichia coli protein of unknown function[END_REF][START_REF] Brown | Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology[END_REF][START_REF] Teplyakov | Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis[END_REF]. The titration of Ec-YjeE with ADP or ATPγS yielded Kds of 4.9 µM and 14.1 µM and ∆H values of -3.6 Kcal/mole and -2.08

Kcal/mole, respectively (Fig. S3C and D and Table 2). This confirms that also Ec-YjeE preferentially binds to ADP rather than ATP and it also correlates with the fact that most small G-proteins preferentially bind to GDP compared to GTP [START_REF] Allali-Hassani | Probing the active site of YjeE: a vital Escherichia coli protein of unknown function[END_REF][START_REF] Cherfils | Regulation of small GTPases by GEFs, GAPs, and GDIs[END_REF]. onto tRNA. The hydrolysis of latter ATP is not needed for the chemistry of the transfer reaction and might therefore play a regulatory role [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. To better understand the need for ATPase activity in bacterial biosynthesis of t 6 A, we measured the hydrolysis of ATP into ADP by YgjD, YeaZ, YjeE, and YrdC using a NADH-coupled ATPase activity assay. Individual YgjD, YeaZ, YjeE and YrdC do not exhibit ATPase activity whereas YgjD exhibits very weak ATPase activity in presence of YeaZ or YjeE (Fig. 3A). However the ATPase activity of the YgjD-YeaZ heterodimer is strongly activated by YjeE and this effect is maximal for a 1:2 YgjD-YeaZ:YjeE stoichiometry.

Kinetic characterization of the ATPase activity of the YgjD-YeaZ-2xYjeE complex against ATP concentration yielded a K m and k cat of 0.644 mM and 0.4 s -1 (Fig. S4), in comparison with reported values of YjeE alone of 1.4 mM and 0.003 s -1 respectively [START_REF] Allali-Hassani | Probing the active site of YjeE: a vital Escherichia coli protein of unknown function[END_REF]. The ATPase activity is not affected by the addition of neither YrdC nor tRNA Lys (Fig. 3A). We observed that YjeE exists in an equilibrium between monomer and dimer (Fig. 6A and Fig. S4C). Consistent with this observation the ATPase activity of YjeE is also proportional to the percentage of monomeric YjeE (Fig. S4C), suggesting monomeric YjeE binds to and is activated by YgjD-YeaZ. In addition, the ATPase activity of YjeE is regulated by the heterodimer YgjD-YeaZ in a cooperative fashion as the reaction velocity was plotted against YjeE using an Allosteric Sigmoidal equation, giving a Hill value of 1.98 (Fig. S4D). The ATP-mediated interaction between YjeE and the heterodimer YgjD-

YeaZ

Prompted by the strong activation of the ATPase activity of YjeE by YgjD-YeaZ, we further investigated the interaction between YgjD-YeaZ and YjeE by ITC and native gel shift experiments. YgjD or YeaZ alone do not bind YjeE (Fig. 1A and Fig. S1D-F), but the YgjD-YeaZ heterodimer displays a clear gel shift when combined with YjeE (Fig. 4A and Fig. S6C). Very importantly, in all our binding experiments we observed interaction between YgjD-YeaZ and YjeE only in presence of ATP and Mg 2+ . ITC titration of YgjD-YeaZ with YjeE revealed heat exchange signals, but the ITC curve is complex and could not be interpreted by a simple binding process, probably due to ATP hydrolysis during the titration (Fig. S5F). We therefore quantified the binding of YjeE to YgjD-YeaZ in presence of non-hydrolysable AMPPNP and Mg 2+ (Fig. S5A).

The resulting binding curve yielded a Kd of 0.6 µM, a ∆H of 10.47 kcal/mol and a 1:1:1 stoichiometry for the YgjD-YeaZ-YjeE complex (Table 1). The 1:1:1 stoichiometry was confirmed by varying the YjeE concentrations in the gel shift experiments (Fig. 4A). We did not observe an interaction between YgjD-YeaZ and YjeE in presence of ADP (Fig. 4B and Fig. S5D andE). We conclude that YjeE forms a complex with YgjD-YeaZ only in presence of ATP and that it contacts both YgjD and YeaZ. [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

The YgjD-YeaZ-YjeE ternary complex is required for biosynthesis of tRNA t 6 A We then investigated the contributions of these proteins to the in vitro biosynthesis of tRNA t 6 A. Overexpressed tRNA Lys was first incubated with YgjD, YeaZ, YjeE and YrdC in presence of ATP, Mg 2+ , L-threonine and bicarbonate.

At the end of the reaction, tRNA was enzymatically digested, the nucleosides were separated by HPLC gel filtration and analyzed by mass spectrometry. As seen in Figure S7, the presence of the modified adenine could clearly be demonstrated (Fig. S7B-D). As expected, all the four proteins are required for the biosynthesis of t 6 A in the assay (Fig. 5). In a second experiment we investigated whether YrdC and YgjD-YeaZ-YjeE contribute independently to the overall synthesis. We therefore first synthesized TCA from a solution contain-ing L-threonine, ATP, bicarbonate and YrdC (results not shown). After the removal of YrdC by ultra-filtration, we were able to reconstitute the biosynthesis of tRNA t 6 A by mixing the eluate with YgjD, YeaZ and YjeE. This conforms previous similar experiments carried out with Qri7, and the B. subtilis orthologs of YgjD, YeaZ and YjeE. It therefore seems that all the t 6 A modification systems use the same overall reaction scheme [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Structural model of the ternary complex YgjD-YeaZ-YjeE in solution.

We were unable to crystallize the YgjD-YeaZ-YjeE complex in presence of ATP or AMPPNP. We performed SAXS experiments in order to obtain structural data of the complexes in solution (Fig. 6, Fig. S8, Table S2). We first analyzed the scattering curves of the individual YgjD, YeaZ, YgjD-YeaZ and YjeE components in presence of ATP and Mg 2+ . Gel-filtration and SAXS data confirmed that the isolated YgjD and YeaZ form homodimers in solution and that YgjD-YeaZ is a heterodimer. As explained in the supplementary data, the X-ray scattering curves of YgjD, YeaZ and YgjD-YeaZ can be satisfactorily described from the crystal structures using the SASREF program (Fig. S8A, S8G and S8C, respectively). The SAXS curve (Fig. S8B) of the mutant YgjD S97E is compatible with a monomer showing that the mutation disrupts the dimer of YgjD. The best fit was obtained by using a model with a slightly widened interface between the N-and C-terminal domains compared to the wild type structure in complex with YeaZ. Gel filtration analysis of YjeE shows it adopts a monomer-dimer equilibrium that shifts towards the dimeric form over time (Fig. 6A and Fig. S4C). By using a HPLC gel filtration step just before the SAXS measurements, we were capable of obtaining data on the separated monomeric and dimeric fractions of YjeE. The calculated scattering curve obtained from a homology model of monomeric Ec-YjeE (see Supplementary materials) is in very good agreement with the scattering curve (Fig. S2D). In the case of the dimer, an envelope was obtained by using the ab initio program GASBOR (Fig. S2F andS2G). Two subunits of Ec-YjeE could be fitted into the envelope, but with only SAXS data we could not propose a precise orientation of the subunits of the dimer. We then used gel filtration coupled to SAXS to follow the formation of the YgjD-YeaZ-YjeE complex in presence of ATP. The gel filtration profile of a mixture of YgjD-YeaZ and YjeE in a 1:4 stoichiometric ratio yielded three peaks that correspond to YgjD-YeaZ-YjeE, homodimeric and monomeric YjeE, respectively (Fig. 6A). Analysis of the same protein mixture in presence of ADP showed no evidence for the formation of a stable ternary YgjD-YeaZ-YjeE complex (Fig. S8D).

The X-ray scattering data revealed that YgjD-YeaZ-YjeE has exactly the same maximal extension than the YgjD-YeaZ dimer (D max =90 Å, Fig. S8H) and that the envelope obtained using GASBOR (Fig. 6B) is compact. These results suggest that YjeE binds at the YgjD-YeaZ interface, rather than forming an extended complex. This interpretation of the SAXS data is compatible with biochemical data showing that only the YgjD-YeaZ heterodimer binds to YjeE and not the respective homodimers. We then attempted to create a structural model compatible with SAXS scattering curves and biochemical data. Since the presence of ATP is mandatory for the formation of the YgjD-YeaZ-YjeE complex, we reasoned that the ATP-binding site of YjeE might be involved in the interaction with YgjD-YeaZ. On the other hand, we noticed that the ADP bound at the YgjD-YeaZ interface makes contacts with both YeaZ and YgjD (Fig. 2C) and that its orientation is inverted compared with the ADP bound to YjeE (Fig. 6C). We therefore constructed a model of the ternary YgjD-YeaZ-YjeE complex using the SASREF program by requiring ADP of YgjD-YeaZ to be bound to YjeE in the same manner as the ADP to YjeE (PDB ID:1HTW). The calculated SAXS curve of the YgjD-YeaZ-YjeE model is in excellent agreement with the experimental scattering curve (Fig. 6B). In this model, YjeE sits on top of the helical bundle formed by α2 and α3 from YgjD and α1 and α2 from YeaZ and the three partners in the complex contribute to the formation of the ATP site. The regions surrounding the ATP site are very well conserved (Fig. 2E and6F). The structure of the YjeE ATP complex is not known and there might exist significant structural rearrangements in the switch regions between the ADP and ATP bound forms.

Exploration of structural data by site directed mutagenesis

Active site mutants of YgjD

The importance for t 6 A activity of the two conserved histidinesthat coordinate a Fe ion in Kae1/Qri7 was already investigated [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. We wanted to analyze the importance of the Mg 2+ ion, by mutating its totally conserved Glu 12 carboxylate ligand (Fig. 5B) into alanine. The crystal structure of YgjD E12A -YeaZ in presence of ATP showed the absence Mg 2+ in the active site. Interestingly, this mutant has ATP bound at the active site compared to ADP for the wild YgjD-YeaZ type although no binding of AMPCPP to YgjD E12A was observed by ITC (Fig. S3J). YgjD probably has a very weak ATPase activity, which is inhibited in the YgjD E12A mutant, explaining why we observed ATP at the active site of the mutant. Moreover, there was no electron density observed for ADP at the YgjD E12A -YeaZ interface, suggesting this site could have a preference for ADP rather than ATP. We further observed that the in vitro t 6 A activity of YgjD E12A is totally abolished (Fig. 5). However the ATPase activity of the ternary complex YgjD E12A -YeaZ-YjeE is not affected by this mutation (Fig. 3B). These data further confirm that YgjD is responsible for the t 6 A coupling reaction and YgjE for ATP hydrolysis.

The interfacial ADP binding site

We wanted to test the pertinence of the ADP bound at the YgjD-YeaZ interface (Fig. 2C) using two mutants, YeaZ R118A and YgjD V85E . The crystal structures of both mutants in the presence of ATP showed no ADP-like electron density at the dimer interface. Nonetheless, YgjD-YeaZ R118A still interacts with YjeE in presence of AMPPNP, as determined by ITC (Table 1 and Fig. S5C).

The ATPase activity of the ternary YgjD V85E -YeaZ-YjeE and YgjD-YeaZ R118A -YjeE complexes remained unaffected (Fig. 3B) and the latter is fully active for the in vitro biosynthesis of t 6 A (Fig. 5A).

The YgjD-YeaZ interface

In order to estimate the importance of heterodimer formation, we generated mutations at the YgjD-YeaZ interface. Ser 97 and Phe 100 of YgjD are both involved in packing of the helices and are surrounded by hydrophobic residues (Fig. 6C). ITC, gel filtration and native gel data showed that YgjD S97E , YgjD S97R and YgjD F100E , are no longer capable of forming homodimers nor heterodimers with YeaZ (Table1 and Fig. S1C for YgjD S97E by ITC and Fig. S6C for YgjD S97E , YgjD S97R and YgjD F100E by native gel shift), suggesting that the same regions of YgjD are involved both for homo-and heterodimer formation. We determined by ITC and native gel shift that a mixture of YgjD S97R and YeaZ is not able to associate with YjeE in presence of AMPPNP or ATP (Fig. S5I and Fig. S6C). None of these variants are capable of activating the ATPase activity upon mixing with YeaZ and YjeE (Fig. 3B), suggesting that the formation of the YgjD-YeaZ heterodimer is mandatory for the ATPase activation of YjeE.

However, the in vitro t 6 A activity YgjD S97E decreased by only ~40% (Fig. 5A and Fig. S7H).

YjeE ATP binding site mutations

YjeE displays structural features typical for GTPase proteins [START_REF] Allali-Hassani | Probing the active site of YjeE: a vital Escherichia coli protein of unknown function[END_REF][START_REF] Brown | Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology[END_REF], characterized by the presence of switch regions that undergo conformational changes upon nucleotide hydrolysis, triggering signal transduction to other proteins or nucleic acids [START_REF] Cherfils | Regulation of small GTPases by GEFs, GAPs, and GDIs[END_REF]. Two such switch regions were identified in the structure of YjeE: switch I that is involved in coordination of the Mg 2+ ion and switch II in proximity to the β-and γ-phosphates of bound nucleotide (Fig. S2E). To further explore the role of YjeE we designed two types of mutations: those with the goal of affecting nucleotide binding and ATPase activity (residues Thr 43 and Glu 108 ) and mutations of the putative switch regions (Trp109 and Try 82 ). Tyr 82 from switch II is in a connection between β3 and β4 strands and should be close to the γ-phosphate of ATP whereas Trp109 from switch I is close to the Mg 2+binding residue Glu 108 and stacks against Tyr82 (Fig. S2F). The ATPγS-or ADP-binding properties of these mutants were quantified by ITC. While YjeE Y82A and YjeE W109A still bind to ATP and ADP with a Kd of 8.9 µM and 5 µM, respectively (Table 2 and Fig. S3E andF), the binding of ADP to both YjeE T43A and YjeE E108A was abrogated (Fig. S3K andS3L). The ATPase activity of the YjeE Y82A has dropped to 40% and that of the other mutants fall below 10% compared to the wild-type YjeE (Fig. 3B). These data strengthen the hy-pothesis that YjeE is the bona fide ATPase activated by the YgjD-YeaZ heterodimer.

We further tested whether these mutants are capable of forming a complex with YgjD-YeaZ. Native gel shift experiments (Fig. 4A) showed that the YjeE T43A and YjeE E108A mutants no longer interact with YgjD-YeaZ while the binding capacity of the switch mutants YjeE Y82A and YjeE W109A remained unaffected. ITC measurements further showed that the binding capacity of YjeE W109A to YgjD-YeaZ is comparable to that of wild type YjeE in presence AMPPNP (Table 1 and Fig. S5B). The X-ray scattering curve of the YgjD-YeaZ-YjeE W109A mixture however is identical to that of the wild type complex, suggesting that its overall structure is not affected (Fig. S8F).

We then wanted to find out if the ATPase activity of YjeE is essential for the in vitro biosynthesis of tRNA t 6 A. The YjeE E108A and YjeE W109A mutants no longer exhibit ATPase activity (Fig. 3B). Interestingly, the in vitro biosynthesis of t 6 A by YjeE W109A remained unaffected while it dropped by 60% for YjeE E108A (Fig. 5A). We conclude that for the in vitro biosynthesis of tRNA t 6 A the formation of the ternary complex YgjD-YeaZ-YjeE seems indispensable but not the ATPase activity.

Deletion of the C-terminal region of YeaZ

As the C-terminus of is not observed in the crystal structure of the YeaZ homodimer but is well-positioned by interaction with YgjD in the crystal structure of the YgjD-YeaZ heterodimer (see further), truncated YeaZ 1-219 was produced to test the contribution of the C-terminal YeaZ 220-231 in formation of heterodimer YgjD-YeaZ. Binding was followed by ITC and native gel shift and demonstrated that YeaZ 1-219 still interacts with YgjD with slightly higher affinity (Kd of 34 nM compared to 122 nM for intact YeaZ) (Fig. S6B, Table 1 andFig S1B). The X-ray scattering curve for the YgjD-YeaZ 1-219 -YjeE complex was identical to that of YgjD-YeaZ-YjeE (Fig. S8E), confirming the Cterminal tail of YeaZ 220-231 is not essential for the formation of the ternary complex.

Discussion

The YgjD-YeaZ heterodimer is essential for activity

The reaction scheme of the biosynthesis of tRNA t 6 A in bacteria is composed of two main steps. In the first half of the reaction, YrdC utilizes ATP, Lthreonine and bicarbonate to synthesize an unstable intermediate threonylcarbamoyladenylate (TCA), whose threonylcarbamoyl-moiety is subsequently transferred onto the adenosine at position 37 of the substrate tRNAs by the cooperative action of three proteins YeaZ, YgjD and YjeE. The yeast mitochondrial ortholog of YgjD, Qri7, is capable of transferring the threonylcarbamoyl-moiety of TCA onto tRNA without assistance of other protein partners [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. This provides strong evidence that YgjD is responsible for the coupling reaction while YeaZ and YjeE assist its action [START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. How exactly YeaZ and YjeE contribute to tRNA t 6 A modification in vivo remains unknown. We here showed that YgjD forms a stable heterodimer with YeaZ, and that this dimer forms a complex with YjeE in presence of ATP, confirming previous studies on the S. thyphimurium orthologs (24). The YgjD-YeaZ interface is very similar to that of the YeaZ and Qri7 homodimers [START_REF] Stelter | A complement to the modern crystallographer's toolbox: caged gadolinium complexes with versatile binding modes[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. Mutations disrupting the YgjD-YeaZ abrogated interaction with YjeE and are largely inactive in the in vitro biosynthesis of tRNA t 6 A. The same dependence upon dimerization of the t 6 A biosynthesis was observed for Qri7 [START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

Interestingly we observed that YgjD alone also forms homodimers but these are unable to bind YjeE and are inactive. The YgjD S97E , YgjD S97R and YgjD F100E mutants neither form homodimers nor heterodimers with YeaZ, suggesting the YgjD homodimer is structurally similar to those of Qri7 and YeaZ. The complementation of E. coli ygjD requires both the ygjD and yeaZ orthologs from B. subtillus, suggesting a species-specific recognition between YgjD by YeaZ (El Yacoubi et al, 2011).

The coupling of TCA to A 37 of cognate tRNA in archaea and yeast is carried out by the KEOPS complex that consists of a linearly arrangement of the Kae1, Bud32, Cgi121 and Pcc1 proteins [START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Pcc1 is required for the in vitro biosynthesis of tRNA t 6 A in both ar-chaea and yeast cytoplasm [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF][START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF]. In the structural model of archaean KEOPS, Pcc1 binds to the N-terminal lobe of Kae1 whereas Bud32 binds to the C-terminal lobe.

Pcc1, although unrelated to YeaZ, forms a complex with Kae1 whose interface is structurally mimicking that of YgjD-YeaZ. From all these data it seems obvious that the binding of a protein partner to the N-terminal lobe of Qri7/Kae1/YgjD is essential for t 6 A activity. This partner subunit (Qri7, Pcc1 or YeaZ) is likely necessary for binding of the tRNA substrate. We were not able to detect the binding of tRNA neither to the YgjD-YeaZ-YjeE complex nor to any of the individual proteins by electrophoretic mobility shift assay (data not shown), meaning that the tRNA binding is too weak to be detected and occurs only transiently.

The ATPase YjeE is strongly activated by YgjD-YeaZ

The essential role of YjeE in the transfer of threonylcarbamoyl-moiety from TCA onto A 37 of tRNA in bacteria remains unknown [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF]. YjeE has a nucleotide-binding fold with a unique P-loop, sharing features with different families of nucleotide hydrolyzing enzymes such as ATPases, kinases and small G-proteins [START_REF] Teplyakov | Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis[END_REF]. Previous two hybrid data showed that YgjD, YeaZ and YjeE form a network and that the interaction between YeaZ and YjeE was mutually exclusive with the formation of the YgjD-YeaZ complex. Our experiments however demonstrate that only the YgjD-YeaZ heterodimer strongly interacts with YjeE and that this interaction is dependent on the presence of ATP/Mg 2+ . In S. typhimurium YjeE was reported to bind to YgjD-YeaZ and to YgjD, both in presence of ADP or ATP [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. Small GTPases switch between GTP and GDP bound forms. GDP is usually tightly bound and GTP hydrolyzed very slowly: small GTPases therefore need the assistance of Nucleotide Exchange Factors (GEFs) that facilitate dissociation of GDP and association of GTPase activating proteins (GAPs). Small G-proteins and regulatory ATPases contain well-conserved peptide segments near the bound nucleotide, called switch regions. Those are the main binding sites for effectors as well as for their GEFs and GAPs, which can discriminate between the GTP and GDP bound forms. From its resem-blance with small GTPases, the crystal structure of Hi-YjeE in complex with ADP suggested that YjeE might work as a molecular switch triggered by ATP hydrolysis [START_REF] Teplyakov | Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis[END_REF]. We showed that Ec-YjeE binds more strongly to ADP then to ATPγS or AMPPNP and that its intrinsic weak ATPase activity is strongly activated by binding to YgjD-YeaZ (Allali-Hassani et al, 2004;[START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF]. Mutations that disrupt YgjD-YeaZ (YgjD S97E , YgjD S97R or YgjD F100E ) are incapable of activating the ATPase activity of YjeE.

The YjeE W109A (in switch I) and YjeE Y82A (in switch II) mutations did not affect ATP or YgjD-YeaZ binding, but they lost their ATPase activity by 100% and 65% respectively. The in vitro t 6 A biosynthesis of YjeE W109A remained intact.

The YjeE T43A and YjeE E108A mutations abrogated ADP-or ATP-binding and hence they were not able to interact with YgjD-YeaZ. The activity of YjeE E108A in biosynthesis of tRNA t 6 A is reduced by more than 60%. The binding of YjeE to YgjD-YeaZ therefore seems to be primordial for tRNA t 6 A modification rather than its ATPase activity. This confirms the observation that the B. subtilis YgjD, YeaZ and YjeE orthologs function in biosynthesis of tRNA t 6 A in absence of ATP [START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF]. The importance of the ATPase activity was studied for the B. subtilis ortholog YdiB. The ATPase inactive K41A mutant had the same negative effect on cell growth than the knockout mutant [START_REF] Karst | The ATPase activity of an 'essential' Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization[END_REF].

ATP-mediated formation of YgjD-YeaZ-YjeE

Our biochemical and structural data suggest that YjeE contacts both YgjD and YeaZ to form a compact heterotrimeric complex. The YgjD-YeaZ interface is surrounded by a well-conserved surface centered on a bound ADP molecule, which we used as an anchor for docking the YjeE protein. The resulting model of the ternary complex brings into light many interesting features. First, the interfacial ADP-binding site is composed of residues from the 3 proteins and may explain the ATPase activating effect of YjeE upon interaction with YgjD-YeaZ. Second, YjeE interacts both with YeaZ and YgjD, explaining the binding specificity for heterodimeric YgjD-YeaZ. Third, in this model YjeE binds to well-conserved surface patches of the YgjD-YeaZ complex. Fourth, YjeE forms the wall of a crater large enough to accommodate the substrate tRNA and at its bottom it positions the catalytic metal site of YgjD. YjeE bound at this position could contribute to the substrate tRNA binding. Our model of the ternary complex fits very well to the SAXS scattering data, but highresolution structural data would be needed to firmly establish the structure.

The rigid superposition created some small steric clashes between Ala 130 and Gln 131 from YjeE and Pro 327 and Arg 328 of YgjD, and between Pro 61 -Thr 64 of YjeE and Gly 120 and Ala 169 of YeaZ, all of which reside in potentially flexible loops. It should be noticed that the YgjD-YeaZ-YjeE complex forms in the presence of ATP but that the complex was modeled using the structure of the YjeE-ADP complex. Small G proteins usually adopt rather different conformations between the GDP and GTP bound forms. It seems therefore plausible that the ATP bound form of YjeE may undergo conformational changes in the switch regions that might relieve the present steric clashes in our model.

The mutations of two residues that contact this ADP molecule (YeaZ R118A and YgjD V85E ) did not confer any effect on the heterodimerization of YgjD and YeaZ, nor on the activation of the ATPase activity of YjeE. The intact t6A biosynthesis but corrupted ATPase activities of the YjeE W109A mutant showed that formation of the ternary YgjD-YeaZ-YjeE complex is primordial.

Conclusion

All organisms use Sua5/YrdC for the biosynthesis of the intermediate TCA, but the bacteria, eukaryotes/archaea and eukaryotic mitochondria developed different strategies for the condensation of threonylcarbamoyl-moiety from TCA onto adenosine at position 37 of tRNA. Although these strategies involve different proteins, they have several points in common. All the tRNA t 6 A modification systems use a closely related threonylcarbamoyl-transferase, Qri7/Kae1/YgjD, sharing a common structure and active site configuration, centered on a very conserved metal cluster. These enzymes all need support from a protein subunit bound to the N-terminal lobe. Although, the structures of these subunits are different for Qri7/Kae1/YgjD, the interfaces between the subunits are almost identical. The most plausible hypothesis about the function of non-catalytic subunits is their involvement in tRNA recognition, but few data are available on tRNA interaction for any of the t 6 A biosynthesis systems.

Kae1 and YgjD further need Bud32/Cgi121/Pcc1/(Gon7) and YeaZ/YjeE to carry out their activity in biosynthesis of t 6 A, respectively. Although Bud32 is annotated as a protein kinase, structural and functional studies and comparison with its paralog RIO kinase led to the hypothesis that Bud32 may act as an ATPase within the KEOPS complex rather than as a protein kinase. RIO kinase plays an essential role in pre-40S ribosomal subunit maturation and its ATP hydrolysis triggers 40S subunit biogenesis. YjeE and Bud32 probably play regulatory roles in t6A tRNA synthesis through their interactions with and/or hydrolysis of ATP. YjeE and Bud32 are unrelated proteins and they probably interact very differently with YgjD and Kae1, respectively, i.e., Bud32 binds to the C-terminal lobe of Kae1 and YjeE probably at the interface. Further studies will have to establish the functional relationships of these proteins within the t 6 A biosynthesis system. The crystal structure of the ternary complex YgjD-YeaZ-YjeE in complex with tRNA will shed light on more complete mechanistic understanding of the transfer of threonylcarbamoyl-moiety from TCA onto A 37 of tRNA.

for purification, followed by successive ion exchange and size exclusion chromatographic steps. The purity of proteins after each purification step was analyzed by SDS-PAGE. After size exclusion chromatography protein fractions with purity greater than 95% in preparative buffer (20 mM Hepes pH 7.5, 200 mM NaCl and 5 mM β-mercaptoethanol) were collected and stored at -80 °C by flash-freezing in liquid nitrogen. YgjD, YeaZ, YrdC, YjeE and their variants were purified by affinity chromatography using NiNTA resins, followed by Q-anion exchange chromatography and size exclusion chromatography.

Isothermal Titration Calorimetric (ITC) assay

ITC measurements for the determination of the thermodynamic parameters of protein binding equilibrium were performed with MicroCaliTC200 (GE Healthcare). For each measurement a total of 20 injections of 2 µl injectant were performed at intervals of 180 seconds under continuous stirring. Normalized area data in kcal/mol of each integrated injected peak were plotted versus the molar ratio of injectant to sample titrated using the Original Plotting Software 7 (MicroCalSoftware, Inc.)

Crystallization

The individually purified YgjD and YeaZ were mixed at a molar ration of 1:1 and the mixture was subjected to size exclusion chromatography in buffer containing 20 mM Hepes pH 7.5, 200 mM NaCl and 5 mM β-mercaptoethanol.

Fractions containing the YgjD/YeaZ heterodimer as visualized by SDS-PAGE gel were pooled and concentrated to ~10.0 mg/ml. Thin plate crystals of the heterodimer YgjD-YeaZ were obtained at 293K by hanging-drop vapor diffusion from a crystallization solution consisting of 4.5 mg/ml YgjD/YeaZ heterodimer, 60 mM Hepes pH 7.5, 100 mM NaCl, 15% PEG4000, 150 mM Lithium Acetate, 2.5 mM ATP-Mg 2+ and 2.5 mM L-threonine. The same purification and crystallization procedures were applied to the mutants. The crystals of the heterodimers YgjD E12A -YeaZ, YgjD V85E -YeaZ and YgjD-YeaZ R118A were obtained under similar conditions. The crystals were cryoprotected in the reservoir solution supplemented with ~25% Ethylene Glycol and flash-cooled and stored in liquid nitrogen until data collection.

Data collection and structure determination

Data collection was carried out on beamlines Proxima1 and Proxima2 at the SOLEIL Synchrotron (Saint-Aubin, France). Data were processed, integrated and scaled with the XDS program package [START_REF] Kabsch | Xds[END_REF]. The isomorphous crystals of the YgjD-YeaZ heterodimer and their mutants, YgjD E12A -YeaZ, YgjD V85E -YeaZ, YgjD-YeaZ R118A , belonged to space group P1 with unit cell parameters of a= 63.62 Å, b= 68.48 Å, c= 87.07 Å, α=109.38°, β=92.66° and γ=117.66°. The structure of the heterodimer YgjD-YeaZ was solved by molecular replacement using the PHASER module from the CCP4 package starting with search models of P. abyssi Kae1 (PDB ID: 2IVP) and E. coli YeaZ (PDB ID: 1OKJ) (Hecker et al, 2007b;[START_REF] Stelter | A complement to the modern crystallographer's toolbox: caged gadolinium complexes with versatile binding modes[END_REF][START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF]. The structural model by built from initial model obtained using Phenix AutoBuild [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF], followed by interactive and manual building using the COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]). The final model was refined to satisfactory values using Refmac5 (Table S1) [START_REF] Winn | Macromolecular TLS refinement in REFMAC at moderate resolutions[END_REF]. Two copies of the YgjD-YeaZ heterodimer were found in an asymmetric unit. ADP or ATP molecules and Mg 2+ ions were modeled into the residual Fo-Fc electron density contoured at 3.0 σ. Additionally, fluorescence measurements on the crystals around the Fe-edge wavelength, confirmed Fe was present in the YgjD structure (data not shown). Finally, water molecules were added manually to the protein model and validated by refinement. Data collection and structure refinement statistics are summarized in Table S1.

Small Angle X-ray Scattering (SAXS) experiment

SAXS experiments were carried out on the SWING beamline at the SO-LEIL synchrotron radiation facility (Saint-Aubin, France). The sample to detector (Aviex CCD) distance was set to 1500 mm, allowing reliable data collection over the momentum transfer range 0.008 Å -1 < q < 0.5 Å -1 with q=4πsinθ/λ where 2θ is the scattering angle and λ the wavelength of the X-rays (λ= 1.0 Å). In order to separate the isolated partners from complexes, SAXS data were collected on solutions eluting from the on line size-exclusion highperformance liquid chromatography (SE-HPLCBio-SEC3 Agilent) column available on SWING and directly connected to the SAXS measuring cell. The purified YgjD, YeaZ, the YgjD-YeaZ heterodimer, YjeE and their mutants were premixed in absence of any added nucleotides or with 1 mM ATP or ADP and 4 mM MgCl 2 . The individual proteins or mixtures of them were injected in the column pre-equilibrated with preparative buffer (20 mM Hepes pH 7.5, 200 mM NaCl and 5 mM β-mercaptoethanol) without addition of any nucleotides or supplemented with 1 mM ATP or ADP and 4 mM MgCl 2 . Flow rate was 200 µL/min, frame duration was 1 s and the dead time between frames was 0.5 s.

For each frame, the protein concentration (between 0.5 and 2 mg/mL at the top of elution peak) was estimated from UV absorption at 280 nm and 295 nm using a spectrometer located immediately upstream of the SAXS measuring cell. Due to the presence of ATP or ADP these measurements give only an order of magnitude of the concentration. Selected identical frames corresponding to each elution peak were averaged. A large number of frames were collected before the void volume and averaged to account for buffer scattering. SAXS data were normalized to the intensity of the incident beam and background (i.e. the elution buffer) subtracted using the programs FoxTrot (courtesy of SWING beamline) and Primus [START_REF] Konarev | ATSAS 2.1, a program package for small-angle scattering data analysis[END_REF][START_REF] Konarev | PRIMUS: a Windows PC-based system for small-angle scattering data analysis[END_REF]. The scattered intensities were displayed on an absolute scale using the scattering by water. The molar mass M of the scattering objects is usually calculated from zero-angle scattered intensity I(0) which is proportional to M and to the concentration. This method cannot be applied in our case because of the uncertainties in concentration. In order to determine unambiguously the oligomeric state of the protein or complex the molar mass was obtained using the macromolecule volume and the SAXS-MoW method available at http://www.if.sc.usp.br/~saxs/. The model of YjeE was built by Phyre 2 using the crystal structure of H. influenzae YjeE (PDB ID: 1HTW) in which the relative coordinates of the ADP, Mg 2+ and water molecules were modelled to YjeE [START_REF] Kelley | Protein structure prediction on the Web: a case study using the Phyre server[END_REF][START_REF] Teplyakov | Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis[END_REF].

The general strategy to analyse the SAXS data consists in using the crystal structures of the partners and in determining the quaternary structure using the program SASREF. The program runs typically twenty times and the model that gives the best adjustment to the experimental curve is chosen. The few residues missing in the Cter parts of the proteins are modelled using the pro-gram MODLOOP. An ultimate adjustment is performed using the program CRYSOL. Only when no information on the relative position of the different partners is available -which is the case of the YjeE dimer and of the complex YgjD-YeaZ-YjeE -a preliminary step is added: the envelope of the macromolecule is deduced from the I(q) curve using the program GASBOR in order to guide the building of the complex.

The structure of the YgjD S97E monomer was extracted from the crystal structure of the heterodimer YgjD-YeaZ determined in this study. The two domains of the monomer were subsequently slightly moved using SASREF in order to fit to the experimental curve. The homodimer YgjD was constructed by superimposition on the crystal structure of YgjD-YeaZ followed by a very slight opening of each monomer. The SAXS curve of the heterodimer YgjD-YeaZ was perfectly described using the crystal structure. The same holds true for the homodimer of YeaZ which was well described using the crystal structure (PDB ID:1OKJ) with the missing C-ter parts built by PHYRE2 and moved using SASREF. The model of YjeE was built by Phyre 2 using the crystal structure of H. influenzae YjeE (PDB ID: 1HTW) in which the relative coordinates of the ADP and Mg 2+ molecules were conserved. This model gives a perfectly adjustment to the experimental curve. The modelization of the ternary complex is described in the main text.

In vitro assay of biosynthesis of tRNA t 6 A and digestion of tRNA

The 25 µg E.coli tRNA Lys was incubated with 2.5 µM wild-type proteins (YgjD, YeaZ, YjeE and YrdC) or their mutants in 80 µl of assay buffer that contained 50 mM Tris pH 7.4, 200 mM NaCl, 10 mM MgCl 2 , 25 mM KCl, 4mM L-threonine, 2 mM ATP and 10 mM sodium bicarbonate. The reactions were proceeded for 90 min at 37 °C and terminated by heating the reaction mixture to 95 °C for 5 min, followed by fast cooling-down in ice water. tRNA was unfolded and the protein precipitates were removed by centrifugation. The tRNA was further enzymatically digested with P1 nuclease, phosphodiesterase and alcaline phosphatase (All products from Sigma), following the instructions of the products and a method developed for analysis of nucleosides by HPLC [START_REF] Gehrke | Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides[END_REF]. The digested nucleosides were applied to HPLC and ▼ Fig. S8 SAXS analysis of protein complexes and partners. A. C. E. F. G. SAXS results for YgjD-dimer, YgjD-YeaZ, YgjD-YeaZ 1-219 -YjeE, YgjD-YeaZ-YjeE W109A and YeaZ-dimer respectively. Comparison of the experimental curve (black dots) with the curve calculated by using CRYSOL (red line) from the model built as described in the text. In all cases the goodness of fit was characterized by the parameter χ given by where N is the number of experimental points, c is a scaling factor, and I calc (q j ) and s(q j ) are the calculated intensity and the experimental error at the scattering vector q j , respectively. B. Saxs curve for the monomer of YgjDS97E. The blue curve is calculated from the crystal structure of YgjD. The red one is obtained by slightly opening the two domains of the protein. Insert: in blue, the crystal structure; in red, the "open" structure. D. HPLC chromatographic profiles of solutions of YgjD-YeaZ alone and in a mixture of large excess of YjeE (4x) in presence of 1.0 mM ADP and 2.0 mM MgCl 2 . H. The distance distribution functions P(r) for YgjD-YeaZ (blue) and YgjD-YeaZ-YjeE (red) deduced from the scattering curves I(q) using the program GNOM. The two functions come to zero for the same value r=D max =90Å.

The biosynthesis of threonylcarbamoyladenylate (TCA) by YrdC/Sua5

Despite the availability of a few X-ray crystal structures of E. coli YrdC and S. tokodaii Sua5 at the start of this thesis, the relationship between structure and function of YrdC/Sua5 family proteins still conceals many poorly understood aspects. In my thesis work, I obtained structural and functional data on the P. abyssi Sua5 and E. coli YrdC that contribute to clarify the reaction scheme of TCA biosynthesis and the possible role of the Sua5 domain.

The conserved catalytic center of YrdC/Sua5

We first confirmed that the E. coli YrdC utilizes bicarbonate, L-threonine and ATP to synthesize the short-lived intermediate threonylcarbamoyladenylate (TCA). We further demonstrate that the Arg52 of E.coli YrdC (equivalent to Arg58 of P. abyssi Sua5), which belongs to the conserved KRSN-tetrad motif in Sua5/YrdC protein, is functionally involved in TCA biosynthesis (data not shown in this manuscript). The structural superposition of P. abyssi Sua5 onto S. tokodaii Sua5 in complex with either AMPPNP [START_REF] Kuratani | Crystal structure of sulfolobus tokodaii sua5 complexed with L-threonine and AMPPNP[END_REF] or TCA [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF][START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF] identified a 20-residue linker (215-235) between N-terminal YrdC and C-terminal Sua5 domains, which was not observed in two crystal structures of S. tokodaii Sua5. The linker completely covers the active site and overlaps with AMPPNP or TCA that were bound in S. tokodaii Sua5 (Fig. 1). The linker of P. abyssi Sua5 is bound to pyrophosphate through hydrogen-bonds with Gly 229 and His 234 . We propose that the linker might play a regulatory role in binding and releasing of reactive intermediates during the reaction cycle. Despite the knowledge of structures of complexes with substrates (L-threonine) and reaction products (TCA, AMP, PPi), the precise reaction mechanism remains unclear.

Sua5 domain as a new fusion partner of YrdC

The C-terminal Sua5 domain adopts a Rossmann-type fold and is related to the GTP-binding proteins HypB [START_REF] Gasper | Structural insights into HypB, a GTP-binding protein that regulates metal binding[END_REF] and Fth [START_REF] Freymann | Functional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP[END_REF], suggesting that the C-terminal Sua5 domain could be involved in binding the nucleotide [START_REF] Agari | X-ray crystal structure of a hypothetical Sua5 protein from Sulfolobus tokodaii strain 7[END_REF]. However, nucleotide binding was demonstrated for the YrdC N-terminal domain and the function of the Sua5 domain remains therefore unclear.

The phylogenetic distribution of the Sua5 domain demands deep analysis since it can be either present or absent in YrdC proteins from the three domains of life. Although the YrdC domain is fused with a Kae1 domain in TobZ and E. HypF, two enzymes that are involved in carbamylation reaction processes [START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF][START_REF] Shomura | Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases[END_REF], no interaction between YrdC and YgjD from E. coli was observed.

The YrdC-domain is covalently linked to a Kae1-domain in a family of enzymes that catalyze related carbamoylation reactions on totally different substrates (TobZ, NodU or HypF). The association of the YrdC-and Kae1domains conceals an interfacial cavity that connects the active sites of these enzymes and likely facilitates the transfer of reactive intermediates from YrdC onto Kae1 (Fig. 2). We were unable to demonstrate an interaction between YrdC/Sua5 and YgjD/Kae1 for any of the t 6 A biosynthesis systems we studied, suggesting that they are either too short-lived or that they are nonexistent. Although the TCA intermediate is unstable, others and we showed that its half-life is sufficiently long to allow dissociation from the active site of YrdC and diffusion to that from Kae1 without the necessity of the formation of a stable complex between the two enzymes.

The yeast KEOPS protein complex in biosynthesis of tRNA t 6 A

The overall contribution of the KEOPS complex to the t 6 A biosynthesis is now well documented. Kae1 accommodates the catalytic center for t 6 A biosynthesis but the role of the other KEOPS subunits is not understood. They are expected to specifically bind substrate tRNA and/or regulate the activity of Kae1 in the threonylcarbamoyl-moiety transfer reaction. In my thesis work I focused on two aspects: first, the fungus-specific subunit Gon7 and its influence on the quaternary structure of the KEOPS complex and the other, the structural details of kinase and ATPase activities of Bud32.

The function of Gon7 and the quaternary structure of yeast KEOPS

At the start of my thesis, there existed no information on the function and structure of fungus-specific Gon7 [START_REF] Srinivasan | The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A[END_REF][START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF], which is essential for the in vitro biosynthesis of tRNA t 6 A. Sequence-based structure predictions indicated that that Gon7 might be totally disordered. The crystal structure of the binary complex Gon7-Pcc1 shows that 60% of Gon7 becomes ordered upon interaction with Pcc1. Gon7 interacts with Pcc1 in the same manner as the Pcc1 subunits in the Pcc1 homodimer from P. furiosus.

The fold of Gon7 is different from that of Pcc1, but it can be considered as a structural mimicry of the latter (Fig. 3).

Pcc1 constitutes a dimerization unit within the archaeal KEOPS complex [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. Our crystal structure of the Gon7-Pcc1 complex showed that Gon7 prevents Pcc1 from homodimerizing and hence the yeast KEOPS is present as a pentamer in solution. The functional significance of these drastically different quaternary structures of the KEOPS complex is not clear. Our SAXS-based model of the quaternary structure of yeast KEOPS proposes a linear molecular architecture shown in Fig. 3. It is similar to the structure proposed for the archaean complex, except that Gon7 replaces the second Pcc1 subunit.

A possible role for the Pcc1 subunit is suggested from data obtained on the archaeal KEOPS complex [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. Archaean Kae1 weakly binds substrate tRNA but the affinity increases for the Kae1-Pcc1 complex, suggesting Pcc1 contributes to tRNA binding. Disruption of the Pcc1 interface in archaeal KEOPS abolishes t 6 A activity showing that the integrity of the dimer of tetramers is required. The docking model of yeast KEOPS shows anticodon of tRNA is accommodated atop the active site of Kae1 and the orientation of A 37 into active site of Kae1 necessiates the direct contribution of Bud32, Pcc1 and Gon7, which is in good aggrement with our tRNA binding tests by KEOPS and its subcomplexes (data not shown in this manuscript).

A Gon7 mutational study of the effects on the t 6 A activity and on tRNA affinity of the KEOPS complex will also be very informative. Remarkably, Gon7 is disordered in solution and only 60% of the sequence becomes structured in complex with Pcc1. The remaining part contains well-conserved sequence stretches that might be important for function.

Bud32: an ATPase or a kinase?

While S. cerevisiae Bud32 and its human ortholog were shown to possess kinase activity [START_REF] Facchin | Functional homology between yeast piD261/Bud32 and human PRPK: both phosphorylate p53 and PRPK partially complements piD261/Bud32 deficiency[END_REF][START_REF] Hecker | Structure of the archaeal Kae1/Bud32 fusion protein MJ1130: a model for the eukaryotic EKC/KEOPS subcomplex[END_REF][START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF] and an in vitro ATPase activity was observed for P. abyssi Bud32 [START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. There is no experimental evidence for the phosphorylation of any of the tRNA t 6 A-modifying proteins and one would not expect to find a kinase to participate in a "static" complex with its substrate proteins. It is therefore unlikely that Bud32 functions as a kinase in the t 6 A biosynthesis pathway. It cannot be excluded that Bud32 is involved in other biological processes where its kinase activity would be required.

Fig. 4 The structure of the binary complex Bud32-Cgi121 in complex with ADP and Mg 2+ . The mutational study that characterized the kinase and ATPase activities is highlighted, with the references as the following: Lys 52 in [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF], Asp 161 in (Facchin et al, 2002; Hecker et al, 2008; Perrochia et al, 2013), D 182 (Ferreira-Cerca et al, 2012; Mao et al, 2008), Ser 187,189 in [START_REF] Facchin | Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life[END_REF].

Mutations on the active site of Bud32 have deleterious effects on cell growth and t 6 A biosynthesis that are similar to those observed for the gene deletion, demonstrating that the function of Bud32 is related to its enzymatic activity (Fig. 4). Previous structural data obtained for archaean Kae1-Bud32 complexes showed that the P-loop of Bud32 was disordered and that no nucleotide analogs were bound to its active site. Structural data obtained on the yeast Bud32-Cgi121 complexes in presence of nucleotides on the contrary, shows that the P-loop adopts a canonical conformation and that the nucleotides are firmly bound to the active site. The ternary complex YgjD-YeaZ-YjeE is required for biosynthesis of tRNA t 6 A E. coli YgjD is responsible for the threonylcarbamoyl-moiety transfer from TCA onto A 37 of tRNA but the role of the other two subunits is not clear. We made considerable progress on elucidating the essential roles of YeaZ and YjeE in tRNA t 6 A modification through structural and functional analysis of the YgjD-YeaZ-YjeE protein network.

The specific recognition between YgjD and YeaZ

The crystal structure of the heterodimer YgjD-YeaZ from S. tyrumrium [START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF] was published when our functional study based on our crystal structure of the heterodimer YgjD-YeaZ from E. coli was in the final phase. The structures of the YgjD-YeaZ heterodimer from E. coli and S.

tyrumrium are almost identical but provided useful information on the potential YjeE binding site (next paragraph). The structures of the YeaZ and Qri7 ho-modimers and the YeaZ-YgjD heterodimer all involve the same interfaces (Fig. 5). The same type of heterodimer interface is present in the Kae1-Pcc1 complex, despite the fact that Pcc1 is totally unrelated to the Kae1/YgjD/Qri7 family. Disruption of the YgjD-YeaZ interface convincingly proved that heterodimer formation is essential for enzymatic activity, providing a strong basis for the strict requirement of YgjD-YeaZ as a unit in genetic complementation study [START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF]. All these structural data demonstrate the necessity of the binding to the N-terminal lobe of Kae1/YgjD/Qri7 for enzymatic activity. Whether this second subunit stabilizes Kae1 and/or is involved in tRNA binding remains to be investigated. The ATPase activity of YjeE is dispensable in t 6 A biosynthesis E. coli YjeE is an essential and conserved ATPase (Allali-Hassani et al,

2004

) that is structurally closely related to a set of P-loop GTPases [START_REF] Brown | Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology[END_REF]. During this work, I found that the low intrinsic ATPase activity of YjeE is activated and regulated by the YgjD-YeaZ heterodimer. Furthermore, YjeE is in slow equilibrium from a monomeric (active) to a dimeric (inactive) form over time (Fig. 6). YjeE has higher binding affinity for ADP than for ATP. The mutations of the Walker A residues (Thr 43 and Glu 108 ) resulted in loss of nucleotide-binding. Mutations of the switch residues (Tyr 82 and Trp 109 ) do not affect the affinity for nucleotides but abrogate the ATPase activity. Compared to the wild type YjeE, the biosynthesis of tRNA t 6 A is not affected by the mutation of Trp 109 . It was therefore possible to uncouple the ATPase activity from t 6 A biosynthesis. More in-depth kinetic measurements will be needed to establish the relationship between ATPase and t 6 A activities of YjeE. ATP binding to YjeE likely will produce conformational changes in the switch regions that will affect/regulate its interaction with YgjD-YeaZ.

-The complex involves contacts between the active site and switch regions of YjeE on the one hand and a highly conserved surface patch around the interfacial ADP binding site at the YgjD-YeaZ interface.

-The proposed binding of YjeE to YgjD-YeaZ would not block the access of the bulky tRNA to the active site of YgjD. The position of YjeE occupied in the ternary complex, would allow it contributing to tRNA binding. The central piece of information missing for a better understanding of the mechanism and function of KEOPS is undoubtedly a high-resolution structure of KEOPS-tRNA substrates. Thanks to the knowledge acquired during this thesis in the preparation of the various protein complexes and the production of large quantities of tRNA, this goal should now be in reachable. Thus, the characterization of assembly of yeast KEOPS and tRNA will serve the ultimate goal to reveal the binding and recognition between KEOPS and tRNA by crystallography. To do so, the preparation of the large quantity of tRNA by in vitro and/or in vivo approaches is an necessary step before large scale crystallization screen. Nontheless, the analysis of folding by gel filtarion and /or Curcular Dichroism and isolation of well folded tRNAs are very important to binding tests and cocrystallization with proteins. However, in absence of a crystal structure, site directed mutagenesis combined with tRNA binding experiments and measurement of enzymatic activities will allow mapping the tRNA binding sites. These data could be combined with high performance modeling of protein nucleic acid complexes. We also hope that constructing an catalytically defective KEOPS complex will help in the crystallization with tRNA substrates. In the same respect, Gon7 is also a potential drug target against fungal pathogens.The interacting interfaces of YgjD/YeaZ/YjeE and Gon7/Pcc1 might be good and specific regions for rational design of small molecules or peptides in order to disrupt their interactions and ultimately, their essential role in cell life. La plupart des ARN de transfert (tRNA) subissent des modifications posttranscriptionnelle nécessaires à leur fonction. La modification t 6 A (N 6threonylcarbamoyladenosine) présente en position 37 des ARNt spécifiques des codons ANN, joue un rôle primordial dans la fidélité de la traduction (appariement correct avec le codon AUG initiateur; prévention des décalages de phase de lecture etc.). La modification t 6 A est catalysée en deux étapes par les protéines de la famille Sua5 /YrdC (aboutissant à la synthèse d'un intermédiaire TCA: threonylcarbamoyladenylate) puis transfert de l'entité Carbamoylthreonine du TCA sur l'ARNt via les protéines du complexe KEOPS chez les eucaryotes et archae ou des protéines YgjD, YeaZ et YjeE chez les bactéries ou encore de la protéine Qri7 dans les mitochondries de levures. Le complexe KEOPS comprend les 4 sous-unités suivantes: Kae1, Bud32, Cgi121 et Pcc1 auxquelles s'ajoutent une 5 ème sous-unité (Gon7) retrouvée uniquement chez la levure. Alors que YgjD est l'homologue bactérien de la protéine Kae1, YeaZ et YjeE n'ont pas d'homologue chez les eucaryotes ni les archées. Jusqu'à présent, les mécanismes catalytiques responsables de la modification t 6 A restent peu connus.

Nous présentons dans cette thèse une série d'études structure-fonction de plusieurs protéines impliquées dans la biosynthèse de la modification t 6 A: Sua5 de P. abyssi; les sous-complexes Bud32-Cgi121 et Gon7-Pcc1 de S. cerevisiae ainsi que le sous-complexe YgjD-YeaZ de E. coli. Les principaux résultats confirment que Sua5/YrdC est l'acteur majeur de la synthèse de l'intermédiaire TCA via son activité pyrophosphatase. Dans la levure, la protéine Gon7, empêche l'homodimérisation de Pcc1 qui ne peut plus induire de dimérisation du complexe entier (alors que c'est le cas chez les archées pour lesquelles Gon7 est absente). La structure du sous-complexe Bud32-Cgi121 de levure fournit des informations essentielles quant à son rôle de Kinase et d'ATPase au sein du complexe KEOPS. Ensemble, ces deux structures Bud32-Cgi121 et Gon7-Pcc1 nous permettent de proposer un modèle pentamérique du complexe KEOPS. Enfin, concernant les protéines bactériennes, nous montrons que l'activité ATPase de YjeE est stimulée par son association au complexe YgjD-YeaZ et que la formation du complexe ternaire YgjD-YeaZ-YjeE a lieu en présence d'ATP. Nous proposons un modèle structural de ce complexe ternaire pouvant expliquer les rôles des protéines YeaZ et YjeE dans la modification t 6 A.

L'ensemble des études structurales abordées dans cette thèse permet donc de mieux comprendre le mécanisme catalytique de la modification t 6 A essentielle et ubiquitaire dans les 3 royaumes de la vie.

Fig. 1

 1 Fig. 1 The role tRNA as an adaptor in genetic information translation. A. Schematic presentation of the central dogma of molecular biology. The arrows represent the genetic informationtransfer. B. The role of tRNAs as adaptor molecules in protein translation (peptide synthesis).

Fig. 5

 5 Fig. 5 The two classes of tRNA synthetases (aaRSs) and the aminoacylation of tRNAs. A. The structural comparison of the two classes of tRNA synthetases. This figure was taken from Dr. Kevin Ahern's lecture material. B. The canonical pathway of aminoacylation of tRNA by the tRNA synthetase (aaRS). The figure was derived from[START_REF] Ibba | Aminoacyl-tRNA synthesis[END_REF].

Fig. 9

 9 Fig. 9 The conservation and phylogenetic distribution of N-terminal YrdC domain (in pink bars) and C-terminal Sua5 domain (in green bars) (courtesy of L. Perrochia, IGM, Orsay).

Fig. 13

 13 Fig. 13The interaction between YgjD, YeaZ and YjeE. A. Genomic synteny of the B. subtilis ydiD, ydiC and ydiB from Genolist database[START_REF] Lechat | GenoList: an integrated environment for comparative analysis of microbial genomes[END_REF]. B. The functional genomic network of Bacillus subtilis ydiB gene from 1133 organisms collected by STRING 9.1 database[START_REF] Franceschini | STRING v9.1: proteinprotein interaction networks, with increased coverage and integration[END_REF]. ydiC, YdiC (229 aa); gcp, putative DNA-binding/iron metalloprotein/AP endonuclease (341 aa); thiL, thiamine monophosphate kinase (327 aa);ydiD,YdiD (152 aa); BL00849, YdiF (641 aa); yxkO, YxkO (283 aa); ydiL, YdiL (244 aa); moaC, molybdenum cofactor biosynthesis protein C (168 aa);ydhE, YdhE (404 aa); yojK, YojK (394 aa). C. The model of the interaction network of YgjD-YeaZ-YjeE in signal transduction in bacteria. YeaZ engages in partner switching in response to an undefined signal, binding with YjeE instead, and free YgjD can then accumulate within the cell. ATP binding or hydrolysis by YjeE is thought to reverse the partner switch, allowing YeaZ to form a complex with YgjD, which is then subjected to proteolysis. The figure C was taken from[START_REF] Msadek | Grasping at shadows: revealing the elusive nature of essential genes[END_REF].

Fig. 14

 14 Fig. 14 The structures of E. coli YrdC, E. coli YciO and S. tokodaii Sua5. A. The structural comparison of YrdC (colored in grey) and YciO (colored in yellow orange). The KRSN tetrad motif and S 139 AN 141 motif in YrdC are colored in red stick and residues at equivalent positions

Fig. 15

 15 Fig.15The structures of Kae1/Qri7/YgjD family proteins. A. The overall fold of P. abyssi Kae1, the domain I and II are represented in blue and red, respectively. B. The overall structure of S. typhimirium YgjD (the Mg 2+ and Zn 2+ ions are represented as green and orange spheres, respectively) and the ribbon is colored according to sequence conservation within the Kae1/Qri7/YgjD family proteins (the conservation scale is given in the key at the bottom). C. The structural superposition of Kae1 (colored in red), YgjD (colored in wheat) and Qri7 (colored in slate) and the two inserts compared to ASKHA family proteins are colored in green and yellow, respectively. D. The conserved Fe-binding motif and ATP-binding residues from P. abyssi Kae1 (residue numbering), YgjD and Qri7.

Fig. 16

 16 Fig. 16 The reaction scheme of TobZ and HypF. A. The biosynthesis of nebramycin by TobZ. The figure was taken from (Parthier et al, 2012). B. The maturation of HypE by HypF. The figure was taken from (Shomura & Higuchi, 2012).

Fig. 17

 17 Fig. 17 The domain conservation and structural similarity of Kae1-and YrdC-like subunits in TobZ and HypF. A. and B. Cartoon representations of KRSN and HxxxH-D motifs in YrdC-like domain and Kae1-like domains of E. coli HypF, respectively. C. and D. Cartoon representations of KRSN and HxxxH-D motifs in YrdC-like domain and Kae1-like domains of S. tenebrarius TobZ, respectively. E. Schematic domain representation of Kae1, Sua5, HypF, TobZ as well as PsV (NC_022098.1). The coloring of the domains, the positions of motifs and the number of residues are roughly proportional to the length of the bar in the representation. F. the overall structure of HypF whose two active sites are represented by two nucleotides. The coloring of the domains of is consistent with thatof HypF in E.

Fig. 18

 18 Fig. 18 Comparison of Pcc1 with nucleic acid-binding KH domains. Ribbons representation of the ssRNA binding KH domain of human Poly(C) binding protein 2 (PDB 2AXY) is shown in green with ssDNA backbone shown in orange. The DNA binding KH3 domain of hnRNP K (PDB 1ZZI) is shown in yellow with ssDNA backbone shown in orange. The RNA hairpin binding KH domain of Nova KH1/KH2 tandem domain (PDB 2ANR) is shown in purple with the RNA backbone shown in orange. Arrow highlights the site of a kink in helix α1 that is a hallmark of the nucleic acid-binding function in KH domains. The figure and accompanying legend were taken from[START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF].

dimer

  YgjD-YeaZ exhibits a large local conformational change on the top of α1 helix, which is in vicinity of the conserved glycine-rich motif 65 GPGXXTGXR[START_REF] Nichols | Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex[END_REF]. However, there is no nucleotide bound in YeaZ in the heterodimer YgjD-YeaZ from S. typhimurium. The interacting interface between YgjD and YeaZ reveals similarity to the interacting interface of Kae1 and Pcc1 or homodimer Qri7 (Fig.20). The incapability of B. subtillus ygjD to complement the E. coli ygjD implicated that the essentiality of YgjD is related to the specific recognition of YeaZ. Furthermore, E. coli ygjD gene could not compensate for the lack of KAE1 in S. cerevisiae, demonstrating that YgjD can't replace Kae1 and interacts with both Pcc1 and Bud32 in the KEOPS protein complex[START_REF] El Yacoubi | A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification[END_REF].

Fig. 20

 20 Fig. 20The comparison of the homo and heterodimer configurations of Qri7/Qri7, YeaZ/YgjD and Kae1/Pcc1 complexes. Bottom panels display close up views of the dimer interfaces shown on top. The figure was adapted from[START_REF] Wan | Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system. Nucleic Acids Res Watson JD, Crick FH[END_REF].

  KEOPS only consists Pcc1, Kae1 and Prpk (ortholog of yeast Bud32), which are encoded by CG42498, CG4933 and CG10673, respectively (Rojas-Benitez et al, 2013). The knockdown of PRPK or KAE1 in Drosophila resulted induction of the unfolded protein response, indicating a correlation between the incomplete entity of KEOPS and an erroneous protein synthesis in the fat body of Drosophila. The human KEOPS is composed of OSGEP, PRPK,

Fig. 22

 22 Fig. 22 Possible mechanisms of action for the KEOPS complex. Two blue lines indicate the chromosome terminus. Thicker lines indicate telomeric DNA. The TG strand is in dark blue and the CA strand is in light blue. In both (A) and (B), the top drawing represents a telomere in the G1 phase of the cell cycle, with minimal TG overhangs and Est2 bound via interaction with Yku.[START_REF] Bianchi | The KEOPS complex: a rosetta stone for telomere regulation?[END_REF][START_REF] Downey | A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator[END_REF]

  To determine the structure of yeast and archaeal Sua5; --To characterize the binding t 6 A substrates to Sua5/YrdC; --To characterize the in vitro biosynthesis of threonylcarbamoyladenylate by Sua5/YrdC --To determine the X-ray crystal structure of YrdC in complex with t 6 A substrates; 3) Biophysical and biochemical characterization of the interaction network of YgjD-YeaZ-YjeE from bacteria --To characterize the binding of nucleotides to YgjD, YeaZ and YjeE; --To characterize the ATPase activity of YgjD or YjeE; --To characterize the in vitro interaction between YgjD and YeaZ; --To determine the X-ray crystal structures of YgjD, YeaZ, YjeE alone or in complex with ATP, ADP and AMP; --To characterize the in vitro interaction network of YgjD-YeaZ-YjeE; --To identify contributions of YgjD, YeaZ and YjeE in t 6 A synthesis through site directed mutagenesis

  Sua5 and P. abyssi Kae1 were produced in E. coli RosettaBlue (DE3)pLysS Competent Cells (Novagen) transformed with pET26b-PAB1302 and pET28a-PAB1159, respectively; S. cerevisiae KEOPS protein complex was produced in E. coli BL21-AI™ Competent Cells (Invitrogen) transformed with pJ241_KEOPS/EKC-WT; S. cerevisiae Kae1-Bud32-Cgi121 ternary complex was produced in E. coli RosettaBlue(DE3)pLysS Competent Cells (Novagen) transformed with pET21a-BC3; S. cerevisiae Kae1-Bud32-6His and Pcc1-Gon7-6His binary complexes and Gon7-strep were produced in E. coli RosettaBlue(DE3)pLysS Competent Cells (Novagen) transformed with 6-pK-Sc-Bu-Cg-a, pET21a-BC1 and pET21a-BC4, respectively; E. coli YgjD, YeaZ, YjeE and YrdC were produced in E. coli BL21(DE3) Competent Cells (Invitrogen) transformed with pET9a-ygjD, pET9a-yeaZ and, pET28a-yjeE and pET26b-yrdC, respectively. T7 polymerase was produced in E. coli BL21(DE3) Competent Cells (Invitrogen) transformed with the plasmid Plasmid pMR-7WT (Generous gift from Professor François Mallet)[START_REF] Arnaud | Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase[END_REF].The transformants listed above were grown in 10 ml 2xYT medium supplemented with appropriate antibiotics and grown overnight at 37 C at a shaking speed of 200 rpm. The precultures were transferred into 800 ml 2xYT medium supplemented with corresponding antibiotics and grown at 37 C at a shaking speed of 200 rpm until the optical density (OD 600 ) of the cell culture reaches 0.6-0.7 for those expressed at 37 C. For those that are expressed at 15 C, cells with an OD 600 of 0.35-0.4 were shifted to 15 C for thermostabilization and kept growing until the OD reaches 0.6-0.7. The induction of the overexpression of each protein was initiated by adding IPTG to a final concentration of 0.5 mM and for 3 hours at 37 °C or overnight at 15 °C. The cell pellets were prepared by centrifugation at 6000g at 8 °C, and were suspended in buffer A (20 mM Tris pH 7.5, 200 mM NaCl and 5 mM βmercaptoethanol) and stored at -20 °C.The cell pellets were defrozen at 37 °C in water bath and lysed by sonication on ice. Cell lysis was centrifuged at 20,000g for 30 min and the supernatant was applied to an Immobilized Metal Affinity Chromatography (IMAC) column that was packed with 3ml Nikel NTA Resin (Qiagen) or Protino ® Ni-IDA Resin (Macherey Nagel) for 6His-tagged proteins or Strep-Tactin resin resin (IBA) for Gon7 and YjeE. The resin bound with proteins was washed and proteins were eluted gradually with appropriate buffers. Subsequently, the fractions containing proteins were collected and further subjected to purification with Cation-or Anion-exchange Chromatography (if necessary for purity) and eventually the Size-Exclusion Chromatography equilibrated with respective buffer. The gel fractions that contain >95% of the target protein were collected and concentrated by centrifugation at 6000g at 8 °C, using ultrafiltration cartridge with the appropriate cutoff. The final concentration of proteins was measured by NanoDrop (Thermo Scientific™) using the predicted extinction coefficient by ProtParam (http://web.expasy.org/protparam/). Pro-teins were immediately used or flash-frozen in liquid nitrogen and stored at -80 °C.

  cerevisiae tRNAIle(UCC) and S. cerevisiae tRNA Asn were ligated 3' adjacent to T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') by PCR. The run-off transcription was carried out by T7 polymerase in a reaction buffer that contains the following chemicals: 20 mM Tris-HCl pH 8.0, 4 mM MgCl 2 , 1mM DTT, 0.5 mM spermidine, 0.01% Triton and 2% PEG8000, 5 mM rNTPs (A, U, C, G), 200 U/ml RiboLockTM RNase Inhibitor (Thermo Scientific) 50 µg DNA template and 1 µM T7 Polymerase. The reaction was undertaken for 4 hours, during which every one hour the reaction buffer was centrifuged for 5 seconds at 1000 g to spin down the pyrophosphate. The reaction was stopped by incubation for 1 min at 95 °C. The DNA template was removed by incubation with 50 U/ml DNase I for 30 min at 37 °C.

  visible spectrophotometer, a differential refractometer, a 7° Low Angle Light Scattering detector, a 90° Right Angle Light Scattering detector and a differential pressure viscometer. The OmniSEC software program was used for the acquisition and analysis of the Viscotek data. BSA was used as standard reference protein of known molecular weight, concentration, refractive index increment (dn/dc=0.185 ml•g -1 ), and intrinsic viscosity to determine the instrument response factors of the detectors for the mobile phase being used.

  suites of Classics, PEGs, PEGsII, MbClass, pHClear, Protein Complex, JSCG+, AmSO4, Anion, Cations, Classics Lite, MbClass, MbClassII, MPD and Nucleix from QIAGEN. All the screening kits were prefilled into Greiner plates (Greiner Bio-One GmbH) and TTP plates (TTP Labtech, UK) by TECAN Genesis and Freedom Evo (TECAN). The crystallizations were carried out as sitting drop by Cartesian Microsys (PROTEIGEN) and Mosquito (TTP Labtech, UK). The further optimization of the initial screening hits was performed on hand-made 96-well or 24-well plates, which are either prepared with TECAN robot or prepared by hand using Eppendorf Pipettes. The optimized crystallization has several variables, for example, the pH of buffer and concentration of the precipitant. The crystallization performed on 96-well plates was monitored by the Rock Maker (Formulatrix, Inc). And the crystallizations performed on 24-well plates were visualized by stereo-microscopes (Nikon). Crystals were cryo-protected by transfer into the crystallization solution provided with progressively higher concentrations of ethylene glycol (EG),

  cleotide and nucleic acid[START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Teplova | The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding[END_REF] and be implicated in the biogenesis of 30S ribosomal particles[START_REF] Kaczanowska | Temperature sensitivity caused by mutant release factor 1 is suppressed by mutations that affect 16S rRNA maturation[END_REF][START_REF] Kaczanowska | The YrdC protein--a putative ribosome maturation factor[END_REF], translational regulation[START_REF] Lin | The Sua5 protein is essential for normal translational regulation in yeast[END_REF], telomere recombination[START_REF] Meng | Sua5p a single-stranded telomeric DNA-binding protein facilitates telomere replication[END_REF] and L-threonycarbamoyladenosine (t 6 A) modification of tRNA[START_REF] Deutsch | The biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside[END_REF][START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Lauhon | Mechanism of N6-Threonylcarbamoyladenonsine (t(6)A) Biosynthesis: Isolation and Characterization of the Intermediate Threonylcarbamoyl-AMP[END_REF][START_REF] Parthier | The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction[END_REF]. The genetic complementation of S. cerevisiae SUA5 by E. coli or M. musculus yrdC suggests at least a role of Sua5 and YrdC in a universally conserved fundamental pathway[START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Lin | The Sua5 protein is essential for normal translational regulation in yeast[END_REF].

  in COOT. The PDB codes of structures of Pa-Kae1, Ec-YrdC, St-Sua5, St-TobZ and Ec-HypF are 2IVP, 1HRU, 2EQA (3AJE, 4E1B), 3VER and 3TSQ, respectively. All the graphic representations of the protein structure were produced with the PyMOL Molecular Graphics System (The PyMOL Molecular Graphics System, 2010), and the electrostatic potential representation and graphs of the ligands coordination were rendered and produced by PDB2PQR (Dolinsky et al, 2004) and with LigPlot + (Laskowski & Swindells, 2011).

Fig 1

 1 Fig 1 Crystal structure of P. abyssi Sua5 (Pa-Sua5) in complex with L-threonine and pyrophosphate. A, Sua5 is composed of a N-terminal YrdC domain (blue) and a C-terminal Sua5 domain (yellow) which are connected by a linker (red). The 2Fc-Fo electron density map for pyrophosphate and Lthreonine is contoured at 1.0 σ. B, The sequence conservation of Sua5 as mapped onto the structure of Pa-Sua5. Sequence conservation scale is goven in the key at the bottom. C, The electrostatic potential surface of the structure of Pa-Sua5. The negatively-and positively-charged regions are represented gradually from red to blue. All the above representations are rendered in the same orientation.

Fig 2

 2 Fig 2 Interaction of ligands with Pa-Sua5. A and B, The coordinations of L-threonine and pyrophosphate. L-threonine and PPi shown in purple sticks and dash lines represent the hydrogen bonds. C, The ITC titration of 20.0 µM Pa-Sua5 with 240.0 µM AMPCPP. The curve is fitted to a one-site model with n, Kd and ΔH of 1.2, 2.1 uM and -1.9 kcal/mol, respectively. D, The titration 0.1 mM PaSua5 with 1.2 mM organic pyrophosphate. The curve is fitted using a simple binding model with n, Kd and ΔH of 0.94, 50 µM and -1.2 kcal/mol, respectively.

  intermediate by the Sua5/YrdC enzyme. Organisms either have a short form of the enzyme made solely of the YrdC-domain or a longer form made of a fusion between a YrdC-and a Sua5-domain. It was shown that the individually expressed YrdC-and Sua5-domains of the long form Pa-Sua5 were inactive (Perrochia, L., Ph. D Thesis, University of Paris-Sud). Mixing of the two domains did not recover the TCA-activity of the intact protein and both domains did not interact in vitro. Ec-YrdC was shown capable of replacing Pa-Sua5 and Sc-Sua5 in t 6 A biosynthesis[START_REF] El Yacoubi | The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA[END_REF][START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF]. The integrity of the long form enzymes is clearly necessary for enzymatic activity but the role of the Sua5 domain remains unclear.

Fig 3

 3 Fig 3 The structural comparison between Pa-Sua5 and St-Sua5. (A) Pa-Sua5 (blue and yellow) and St-Sua5 (beige) YrdC domains are superposed (PDB ID: 3AJE and 4E1B) shows that the linker (colored in red) makes a spatial clash with either the AMPPNP or TCA (both in sticks) in the active site of St-Sua5; (B) The 2Fc-Fo electron density map around the linker in Pa-Sua5 is contoured at 1.0 σ; (C) The structural superposition of AMPNP/TCA, L-threonine and pyrophosphate that are bound in St-Sua5 and Pa-Sua5, respectively. The conserved KRSN tetrad (blue sticks) in Pa-Sua5 is labeled; The Sua5 domain has no close structural analogs in the PDB database. One of the helical surfaces of Sua5-domain interacts intensively with the loops surrounding the active site pocket of YrdC. Interestingly the Sua5 domain residues facing the YrdC domain are very well conserved and form a roof above the YrdC active site pocket. This suggests that this surface of the Sua5-domain participates in substrate

Fig. S2

 S2 Fig. S2 The structural alignment of the apo-and complex structures of Pa-Sua5. A, The YrdC domain, Sua5 domain and the linker in the L-threonine-pyrophosphate are colored in blue, yellow and red, respectively, and the L-threonine bound Pa-Sua5 is colored in pale-yellow. The 2Fo-Fc electron density of L-threonine (blue and pale-yellow sticks), pyrophosphate (red sticks) and two water molecules (shown as spheres) is contoured at 1.0 σ. B, The 2Fo-Fc electron density of pyrophosphate and L-threonine bound to Pa-Sua5. C, The electron density of two water molecules (shown in spheres) and L-threonine in the apo-structure. D, The active site cavity is made of residues mainly from the YrdC domain and the linker. E, The opening atop the pyrophosphate

Fig. 1

 1 Fig.1Crystal structure of the Gon7/Pcc1 complex from yeast. A. The cartoon representation of the Gon7 (colored in green) and Pcc1 (colored in purple). The missing regions in the structure of Gon7 are represented by green dash lines. B. and C. The structural superposition of P. furiosus Pcc1 homodimer (colored in light purple) onto the Gon7/Pcc1 complex. D. The Gon7/Pcc1 heterotetramer type 1 in which Gon7 functions as a homodimerization module. E. The Gon7/Pcc1 heterotetramer type2 in which both Gon7 and Pcc1 contribute to the interaction.

Fig. 2

 2 Fig. 2 Cartoon representations of the crystal structure of the Bud32/Cgi121 complex from yeast. Cgi121 (colored in yellow) binds to the N-lobe of Bud32 (colored in blue) and ADP (thick sticks) and Mg 2+ are bound in the active site between the two lobes of Bud32. The residues whose mutation abolished the kinase and ATPase activities of Bud32 are highlighted in sticks and labeled. The dotted red line denotes the missing loop between Arg 58 and Leu 68 .

Fig. 3

 3 Fig. 3 The structural model of yeast KEOPS. A. The structures of Gon7 (green), Pcc1 (purple), Kae1 (red), Bud32 (blue) and Cgi121 (yellow) are represented. The ATP and ADP moieties in Kae1 and Bud32 are shown in spheres. B. The electrostatic potential surface. The potential is given with the negative (red) and positive (blue) contour levels in the range from -5.0 to +5.0 kBT, respectively. C. The sequence conservation projected onto the surface as reported by ConSurf (http://consurf.tau.ac.il/). The least conserved residues and highest conserved residues are scaled from cyan to dark purple. All the three structure representations are rendered in the same orientation.

Fig. 3 .

 3 Fig. 3. Like archaean KEOPS, yeast KEOPS forms a linear arrangement of subunits, with Gon7 at one end and Cgi121 at the other. The active site of Kae1, centered on the conserved metal cluster and containing the ATP binding site is at the bottom of a crater whose walls are made of Kae1, Bud32, Pcc1 and Gon7.

Fig. 4

 4 Fig. 4 The determination of the quaternary structure of yeast KEOPS. A. The gel filtration profile and molecular weight extracted by SEC-MALLS. B. The fitting of the yeast KEOPS model (red line) to the SAXS experimental curves (black dots). The missing regions of Gon7 were added by modeling with Bunch program.

Fig. S6

 S6 Fig. S6 The fitting of two types of heterotetramer of Pcc1-Gon7 retrieved from the crystal packing into the SAXS ab initio l envelope. A.The Gasbor envelop obtained from the scattering curves of Pcc1-Gon7. B. The fitting of tetramer model 1 and C. the possible formation of a dimer of the heteropentameric KEOPS as mediated by the Pcc1-Gon7 tetramer type 1. D. The fitting of tetramer type 2 and E. The Pcc1-Gon7 tetramer type 2 prevents KEOPS from forming a dimer of the heteropentamer.

Fig. 2

 2 Fig. 2 The crystal structure of the heterodimer YgjD-YeaZ in complex with two molecules of ADP. A. Structural superposition of the YeaZ homodimer (gold) onto the YgjD-YeaZ heterodimer (red and blue). For clarity only one subunit ot the YeaZ dimer is represented). B. Conformational change of the loop 29 LCPREHT 35 (linking β3 and α1) of YeaZ upon its dimerization with YgjD. C. and D. Fo-Fc difference maps contoured at 3.0 σ for ADP molecules bound at the YgjD-YeaZ interface (C) and the active site of YgjD (D). Residues important for the interaction are represented in sticks. E. The sequence conservation projected onto the surface of the YgjD-YeaZ heterodimer. The conservation is scaled and represented by colors with cyan for variable and raspberry for conserved. F. The electrostatic potential surface of the YgjD-YeaZ heterodimer (red negatively and blue positively charged); back and front views are represented, front views (top) are in the same orientation as for views A. G. and H. repre-

  . A similar conformational change was observed for a comparable loop in YeaZ in the St-YgjD-YeaZ heterodimer which did not have any nucleotide bound at the inter-)aminoethane molecule bound which partially overlaps with the phosphate groups of ADP at the Ec-YgjD-YeaZ dimer interface.

Fig. 3

 3 Fig. 3 The ATPase activities of YgjD, YeaZ, YjeE and YrdC, and their mutants. A. ATPase activity of different combinations of wild type proteins. 2xYjeE denotes a molar ratio of YjeE of 2. B. ATPase activity of different combinations of mutants. Null denotes absence of a chemical. 2xYjeE was subjected to guarantee a 1:1 molar ratio of YjeE monomer to YgjD/YeaZ mixture. The bars represent the standard deviations from triplicates.

Fig. 4

 4 Fig. 4 Native agarose gel shift analyses of the interaction between the YgjD-YeaZ heterodimer and YjeE. A. Interaction analyses in presence of ATP and MgCl 2 : lanes 1-6, analyses of YgjD-YeaZ at various stoichiometries of YjeE. lanes 7-12: analyses of YgjD-YeaZ in combinations with various mutants YjeE. B. Interaction analyses of ADP and MgCl 2 . lanes 1-3 analysis of YgjD-YeaZ with wild-type YjeE; lanes 4-6, 3 analysis of YgjD-YeaZ with YjeE W109A mutant.

Fig. 5

 5 Fig. 5 The in vitro biosynthesis of tRNA t 6 A by various combinations of YgjD, YeaZ, YjeE and YrdC proteins. A. Activities are normalized and represented as percentage of the wild type protein activity. TCA denotes threonylcarbamoyladenylate that was produced by YrdC in presence of ATP, L-threonine and bicarbonate (explained in SM). B. Structural superposition of the catalytic residues of the YgjD/Kae1/Qri7 family whose mutations resulted in the abrogation of the biosynthesis of t 6 A. The structure of Sc-Kae1 was modelled by Phyre 2 . ATP was taken from the crystal structure of the YgjD E12A -YeaZ heterodimer. The Mg bound at this indicated position is only observed in the crystal structure of Ec-YgjD. *denotes this study and the superscript numbers denotes the references: 24, (Srinivasan et al, 2011); 14, (Wan et al, 2013); 12,[START_REF] Perrochia | Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification[END_REF].

Fig. 6

 6 Fig. 6 SAXS data and model of the ternary YgjD-YeaZ-YjeE complex. A. The superposition of the gelfiltration profiles of YgjD-YeaZ-YjeE, YgjD-YeaZ and YjeE (4x in molar ratio) in presence of 1.0 mM ATP and 2.0 mM MgCl 2 . B. SAXS scattering curves of the YgjD-YeaZ-YjeE complex. The red line is the calculated scattering curve for the model represented in C; the insert presents the structural model embedded in the most typical envelope extracted from the SAXS curve using the ab initio program GASBOR. C. The cartoon representation of the YgjD-YeaZ-YjeE complex, the interfacial ADP is shown as sticks and interface mutation sites of YgjD are indicated in yellow spheres. D. Comparison of the binding orientations of the ADP from the ternary complex YgjD-YeaZ-YjeE and the ADP (indicated by a cyan arrow) as present in the crystal structure of Hi-YjeE. E. and F. the sequence conservation and electrostatic potential projected on the surface of the YgjD-YeaZ-YjeE complex, adopting same orientation as in C. The scales of the conservation and charge distribution are same as in Fig. 2.

▼

  Fig. S4 The kinetic characterization of the ATPase activity of the ternary YgjD-YeaZ-YjeE complex. A. Calibration curve for consumption of the NADH by ADP, the calculated slope is -0.002421 µmol -1 . B. Kinetics of ATP hydrolysis into ADP by YgjD-YeaZ-2YjeE as a function of ATP concentration. C. The correlation between the ATPase activity (black bars) and YjeE monomeric portion (grey bars). The latter was determined by integration of peaks from the size exclusion chromatogram (not shown). D. The allosteric regulation of the ATPase activity of YjeE by the heterodimer YgjD-YeaZ. The ATPase activity was measured as a function of concentration of YjeE in presence of 2 µM heterodimer YgjD-YeaZ and 4 mM ATP. The data was plotted using allosteric sigmoidal equation (GraphPad).

Fig

  Fig. S5 ITC analysis of the interaction between the heterodimer YgjD-YeaZ and YjeE. A. The titration of YgjD-YeaZ (23 µM) with YjeE (270 µM) in presence of 1.0 mM AMPPNP and MgCl 2 . B. The titration of YgjD-YeaZ (32 µM) with YjeE (380 µM) in presence of 1.0 mM AMPPNP and MgCl 2 . C. The titration of YgjD-YeaZ R118A (40 µM) with YjeE (480 µM) in presence of 1.0 mM AMPPNP and MgCl 2 . D. The titration of YgjD-YeaZ (20 µM) with YjeE (240 µM). E. The titration of YgjD-YeaZ (20 µM) with YjeE (240 µM) in presence of 1.0 mM ADP and MgCl 2 . F. The titration of YgjD-YeaZ (20 µM) with YjeE (240 µM) in presence of 1.0 mM ATP and MgCl 2 . G. The titration of YgjD-YeaZ (23 µM) with YjeE E108A (270 µM) in presence of 1.0 mM AMPPNP and MgCl 2 . H. The titration of YgjD-YeaZ (30 µM) with YjeE T43A (340 µM) in presence of 1.0 mM AMPPNP and MgCl 2 . I. The titration of YgjD S97E /YeaZ (30 µM) with YjeE (360 µM) in presence of 1.0 mM AMPPNP and MgCl 2 .

Fig. S6

 S6 Fig. S6 The characterization of YgjD-YeaZ protein-protein interaction. A. The determination of oligomeric state in solution of YgjD S97R and YgjD F100E by size exclusion chromatography. B. The interaction of the YeaZ 1-219 deletion mutant and YgjD by native gel shift. C. Native gel shift analysis of the effect of the mutations on Ser 97 and Phe 100 of YgjD on its association with YeaZ and YeaZ/YjeE in presence of 1.0 mM ATP and 2.0 µM MgCl 2 .

Fig. 1

 1 Fig. 1 The structural comparison of P. abyssi Sua5, S. tokodaii Sua5, E. coli YrdC (colored in orange) and E. coli Ycoi (colored in green). A. The structural superposition of Sua5 from P. abyssi and S. tokodaii. The linker between YrdC domain and Sua5 domain in the crystal structure of P. abyssi Sua5 is highlighted in red. B. the structural resemblance of E. coli Ycio to the YrdC domain of P. abyssi Sua5. C. The structural comparison between YrdC and YciO from E. coli. The KRSN-tetrad motif is absent in E. coli YciO. The shaded highlights all the ligands in the crystal structures of Sua5 and delineates a proposed mechanism for TCA biosynthesis by Sua5. The red dash denotes the rearrangement of the linker.

Fig. 2

 2 Fig. 2 The structure of TobZ and sequence organization of Kae1, Sua5 and ps-2314 (gi|16607424) from Pandoravirus salinus. The colors correspond in all the representation. The superposition of Kae1 and Sua5 to the Kae1-like domain and YrdC-like domain resulted in a model that exhibits large steric clashes between the Kae1 domain of S. tenebrarius TobZ and Sua5 domain of P. abyssi Sua5.

Fig. 3

 3 Fig. 3 Docking model for KEOPS-tRNA complex by PatchDock. The KEOPS subunits are colored according to the key. The A 37 of tRNA and active site (H141, H145 and D344) of Kae1 are highlighted and denoted.

  Kae1 and Cgi121 seem to have antagonistic effects on the conformation of the Bud32 active site and it remains an open question how the active site of Bud32 might be structured in the complete KEOPS complex. Neither kinase activity nor ATPase activity is chemically required for the t 6 A biosynthesis, and the function of Bud32 must therefore be sought elsewhere. The reconstitution of the complete KEOPS complex hinted towards a role of Bud32 in tRNA binding. The very conserved and positively charged C-terminal helix of Bud32 lies at the entrance of the Kae1 active site and therefore likely participates to tRNA binding. The precise conformation of Bud32 within the KEOPS complex might be governed by ATP binding and hydrolysis. The latter hypothesis was inspired by recent data obtained for the closely related kinase Rio 2, whose ATPase activity was shown to regulate binding to preribosomal particles.

Fig. 5

 5 Fig.5The structural comparison of homodimers YgjD, YeaZ, Qri7, heterodimer YgjD-YeaZ and binary complex Kae1-Pcc1. The interacting interfaces are highlighted in circles

Fig. 6

 6 Fig.6The binding mode of ADP in E. coli YjeE as modeled from crystal structure of H. influenza YjeE[START_REF] Teplyakov | Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis[END_REF] and the transition of YjeE from monomeric form (cartoon representation) to dimeric form (Cartoon in an envelope obtained by SAXS experiment).

Fig. 7

 7 Fig. 7 A structural scenario for the ATP-mediated formation of the ternary YgjD-YeaZ-YjeE complex. The shaded area in YjeE-ATP representation indicates the conformational change of YjeE upon binding of ATP.However, we should bear in mind that the model is based on structures in complex with ADP and that the ternary complex only forms in presence of ATP. The binding of ATP could provoke conformational changes in the switch regions of YjeE which will affect its interaction with YgjD-YeaZ.

  The specific recognition between YeaZ and YgjD is realized by the two conserved α helices from either YgjD or YeaZ. The crystal structure provides for the first time an evidence that an atypical nucleotide-binding site is created in YeaZ upon its interaction with a binding partner. The heterodimer YgjD-YeaZ has been identified as an activator and regulator of the intrinsically week ATPase YjeE. A structural model of a ternary complex YgjD-YeaZ-YjeE is presented in this thesis and it explains how YgjD, YeaZ and YjeE form an ATP-mediated interaction network. It's concluded that YgjD accommodate the catalytic center for transfer of threonylcarbamoyl-moiety onto A 37 of tRNA while YeaZ and YjeE likely play a role in specific binding and correct orientation of A 37 of tRNA into the catalytic center in YgjD. A high-resolution structure of YgjD/YeaZ/YjeE-tRNA complex will certainly reveal not only how the YgjD, YeaZ and YjeE assemble but also the mechanistic binding and recognition of tRNA by YgjD/YeaZ/YjeE ternary complex. The strategy is similar to that proposed for yeast KEOPS.Though the in vitro t 6 A biosynthesis assay doesn't require a direct interaction between YrdC and YgjD/YeaZ/YjeE, the specific binding between YrdC and tRNA is however worth of being explored. Due to the instability of TCA, an association of YrdC with YgjD/YeaZ/YjeE and tRNA facilitates the release and transfer of TCA from YrdC to YgjD. A FRET-based experiment could sensitively monitor the binding of tRNA and association of the protein-tRNA complex when all the interactions occur transiently and are too weak to detect by EMSA experiments. The role of the small YjeE ATPase could be further clarified by obtaining crystals of its complex with ATP or non-hydrolysable ATP analogs. These crystal structures should provide information on the conformational rearrangements upon ATP turn over and explain importance of the conformational change in governing the association of proteins. A premilinary work on crystallization of YjeE has yielded one crystallization condition in which YjeE crystallizes, The crystals diffracted up to 4.2 Å and data shows two molecules of YjeE are present in one asymmetrical unit. Future work could be invested on optimizing the screened crystallization condition and determining the crystal structure of YjeE by molecular replacement. Hopefully, an ATP-bound form structure of YjeE could be determined.In sum, the glitches in maintaining translational fidelity lead to erroneous protein synthesis, resulting in a wide range of lethal phenotypes. However, it necessitates more thorough studies to attribute the roles of Sua5/YrdC, KEOPS, YgjD/YeaZ/YjeE in tRNA t 6 A modification to their essentialities in cell life. Meanwhile, the roles of Sua5 and KEOPS in maintaining the telomere length in yeast need to be further investigated and established. The striking cell morphological changes in ygjD/yeaZ/yjeE temperature-sensitive mutants imply a dominant role the interaction network of YgjD-YeaZ-YjeE in cell wall synthesis. Take a step further, the bacteria-specific YeaZ and YjeE are two promising drug targets for their essentiality to cell life of bacterial pathogens.

  . Structural Basis of the Biosynthesis of the tRNA N6 - threonylcarbamoyladenosine. (Direction: H. van Tilbeurgh et B. Collinet)

Table 2

 2 Summary of studies on mutations affecting KEOPS complex formation and catalytic

	activity. 2008); 2011b); 5 , (Downey et al, 2006); 1 , (Mao et al, 2008); 2 , (Facchin et al, 2002); 6 ,(Kisseleva-Romanova et al, 2006b); 3 , (Lopreiato et al, 2004); 7 ,(Daugeron et al, 4 , (Hecker et al, 8 , (Srinivasan et al, 2011); 9 , (Wan et al, 2013) ; N.D., Not Determined. (Superscript
	numbers refer to the references)

Mutation Growth Phenotype Mutation position Kae1 Null 1,4,7,8 Severe Deficiency in t 6 A N.D H141A 1,8 Severe Deficiency in t 6 A Metal-binding H145A 1,8 Severe Deficiency in t 6 A Metal-binding D344A 1 Severe N.D Metal-binding K27A 1 Severe ATP-binding G190E 1 Severe ATP-binding E292R 1,4 Severe Short telomere; No interaction with Bud32

							Kae1/Bud32 interface
		E295K	1,4	Severe	Kae1/Bud32 interface
		I123R	1		Severe	Kae1/Pcc1 interface
		R126A T377E 1 1	Severe Severe	N.D	Kae1/Pcc1 interface C-terminal tail
		1-364	1		Severe	C-terminal tail
		Null	1,4		Severe	Short telomere	N.D
		D161A	1,2,3,8	Moderate	Short telomere; No kinase activity	Catalytic center
	Bud32	N166A K57E/R72E 1 1-251 1	1,4	Moderate Severe Intermediate	Short telomere No interaction with Kae1 N.D	Catalytic center Bud32/Kae1 interface C-terminal tail
		S187A/S189A	2	N.D.	Reduced Kinase activity	Autophosphorylation site
		K52A	2,5

Severe Short telomere; No kinase activity Catalytic center Cgi121 Null 1 Severe N.D N.D L172E/A176R 1 Intermediate Bud32/Cgi121 interface Pcc1 Null 1,6 Severe Transcriptional misregulation A83R

					1	Intermediate		Pcc1/Kae1 interface
		T79R	1	Severe	N.D	Pcc1 dimer interface
		A83R	1	Severe		Pcc1 dimer interface
	Gon7	Null	5,9	Severe	Short telomere; Deficiency in t	6 A	N.D

Table 1 The X-ray data collection and structural model refinement statistics

 1

	Dataset		Complex-structure	Apo-structure
	Wavelength (Å)		0.98011	0.97857
	Composition		Pa-Sua5-Threonine-PPi	Pa-Sua5-Threonine
	Ligands		4 L-threonine (THR) and	4 L-threonine (THR)
			4 Pyrophosphate (PPi)
	Resolution range (Å)	49.03 -2.617 (2.711 -2.617)	48.78 -2.86 (2.958 -2.856)
	Space group		C2	C2
	Cell	a, b, c (Å)	150.4, 144.1, 103.7	152.2, 142.4, 103.9
	Dimensions	α, β, γ (º)	90, 103.1, 90	90, 101.8, 90
	Unique reflections	64448 (6230)	49930 (4795)
	Completeness (%)	99.44 (96.35)	99.29 (95.23)
	Mean I/sigma(I)		14.29 (2.36)	10.82 (2.21)
	R-factor/ R-free		0.1839/0.2249	0.1924/ 0.2565
	Protein residues	1367	1365
	RMS(bonds)(Å)		0.017	0.024
	RMS(angles)(°)		1.8	1.79
		-favored 97	98
	Ramachandran plot (%)	-allowed 2.7	1.48
		-outliers 0.3	0.52
	Average B-factor	52.4	68.5

Table 1

 1 Data collection and refinement statistics of binary complexes Bud32-Cgi121 and Gon-Pcc1. R sym =Σ h Σ i ⎮I hi -<I h >⎮/Σ h Σ i I hi , were I hi is the ith observation of the reflection h, while <I h > is the mean intensity of reflection h.

	Data collection		Bud32-Cgi121 complex		Gon7-Pcc1 complex
				Apo form	AMPPn*	ADP	AMP	Se-Met	Native
	Wavelength (Å)	0.97918	0.98011	0.97911	0.97911	0.97895	0.98010
	Resolution (Å)	43.20-2.95 (3.06-2.95)	43.71-2.0 (2.07-1.99)	43.62-1.95 (2.02-1.95)	44.01-1.67 (1.72-1.66)	43.53-2.99 (3.10-2.99)	44.28-2.44 (2.53-2.44)
	Space group		I 41	I 41	I 41	I 41	C 121	C 121
		a (Å)		111.54	112.93	113.01	113.89	79.10	80.73
	Cell dimension	b (Å) c (Å) α (°) β (°)		111.54 86.42 90.00 90.00	112.93 87.23 90.00 90.00	113.01 86.35 90.00 90.00	113.89 87.41 90.00 90.00	102.50 88.80 90.00 111.80	104.39 89.89 90.00 111.55
		γ (°)		90.00	90.00	90.00	90.00	90.00	90.00
	Tot. reflections	51370	283335	236236	236981	51149	94329
	Uni. reflections	11205	37120(3660)	39488(3892) 64291(6337) 13327(1325) 25517(2428)
	Completeness	99.8(99.1)	99.79(98.10)	99.89(98.96) 99.41(98.46) 99.54(97.50)	98.9(94.59)
	Mean I/sigma(I)	14.32(2.52)	27.29(5.54)	23.72(6.87)	18.48(3.01)	8.08(2.21)	14.69(2.23)
	Rmeas (%)		12.4(74.6)	4.9 (40.1)	5.4 (26.9)	4.5(49.1)	11.2(46.7)	7.6(58.5)
	CC(1/2) (%)		98.5(82.0)	99.8(98.8)	99.9(97.4)	99.8(86.4)	99.54(97.50)	99.9(87.8)
	Refinement
	Rfactor (%)		21.1	18.3	17.9	19.3	25.1	22.5
	Rfree (%)		28.7	22.2	20.9	21.3	30.8	26.9
	Total atoms		2971	3460	3548	3423	3078	3204
	Average B (Å	2 )	25.70	34.0	31.0	29.0	66.0	64.0
	RMSD from standard stereochemistry
	Bond length (Å)	0.010	0.010	0.005	0.008	0.011	0.014
	Bond angles (°)	1.28	1.29	0.98	1.08	1.48	1.70
	Ramachandran plot statistics
	Favoured (%)	90	98	97	98	98.3	98.4
	Allowed (%)		10	2	3	2	1.7	1.6
	Disallowed (%)	0	0	0	0	0	0
	PDB code		4WWA	4WW5	4WW9	4WW7	4WX8	4WXA
	Values in parentheses are for highest resolution shell.

a b Rfactor =Σ ||Fo| --|Fc|| / |Fo|. Rfree was calculated with a small fraction (5%) of randomly selected reflections.

* The moiety of AMPPNP without γ-phosphate

The ATP-mediated formation of the YgjD-YeaZ-YjeE complex is required for the biosynthesis of tRNA t 6 A in E. coli

	Wenhua Zhang, Bruno Collinet*, Dominique Durand and Herman van Tilbeurgh*
	Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619,
	CNRS, Bâtiment 430, Université de Paris-Sud 11, 91405 Orsay Cedex,
	France
	* To whom correspondence should be addressed:
	Tel: +33 1 69 15 31 55; Fax: +33 1 69 85 37 15; Email: herman.van-
	tilbeurgh@u-psud.fr
	Correspondence may also be addressed to Bruno Collinet. Tel: +33 1 69 15
	79 68; Fax: +33 1 69 85 37 15; Email: bruno.collinet@u-psud.fr
	(To be submitted)

Table 2

 2 The thermodynamic parameters for the binding of nucleotides to YgjD, YeaZ, YgjD-YeaZ, YjeE and the mutants measured by ITC. The corresponding thermograms are presented in Fig.S3. a , in presence of 500 uM MgCl 2 ;

	Titrant (uM)			Injectant (uM)	Stoichiome-try (n)	Kd (µM)	∆H (kcal/mol)	∆S (kcal/mol/ deg)
	YgjD (30)	a			AMPCPP (360)	2.07±0.024	1.62±0.088	2.45±0.045	34.9
	YgjD-YeaZ (30) AMPCPP (360)	1.41±0.062	0.70±0.011	0.91±0.066	31.2
	YjeE (57)	a			ATPγS (680)	a	1.06±0.027	20.41±1.32	-2.07±0.067	15.4
	YjeE (50)	a			ADP (600)	a		0.86±0.026	6.54±0.053	-3.94±0.159	10.5
	YjeE	Y82A (28)	a		ATPγS (350)	a	1.05±0.154	8.90±0.013	-1.26±0.297	19.0
	YjeE	W109A (41)	a	ADP (492)	a		0.82±0.038	4.83±0.022	-5.19±0.327	6.93

factors 34.4 29.10 33.24 38.13 RMS (bonds) 0.016 0.0151 0.0105 0.0159 RMS (angles)

	Dataset	YgjD(ADP) /YeaZ(ADP)	YgjD E12A (ATP) /YeaZ	YgjD(ATP) /YeaZ R118A	YgjD V85E (ATP) /YeaZ
	Data Collection
	Wavelength (Å)	0.98011	0.97857	1.2823	0.9786
	Space group	P1	P1	P1	P1
		a, b, c (Å) 63.62, 68.48, 87.07	63.27 68.07 87.13	63.43 69.28 87.35	63.69 68.08 87.34
	Cell	α, β, γ (º) 109.38, 92.66, 117.66	109.3 92.98 117.23	117.7 108.91 91.17	109.31 92.56 117.94
	Resolution	49.38--2.25 (2.33--	49.04--2.33 (2.42--2.33) a 50.0 --2.8 (2.9--2.7) a 42.62--2.23 (2.31--
			2.25) a			2.23) a
	Rmeas (%)	10.4 (57.7)	25.0 (47.5) b	29.2 (53.6)	8.0 (54.2)
	CC1/2 (%)	99.5 (74.7)	97.9 (87.7) b	84.3 (78.7)	92.4 (65.7)
	<I/σ(I)>	11.00 (2.46)	9.77 (2.98) b	6.24 (2.34)	12.21 (2.16)
	Complete (%)	97.93 (95.69)	98.09 (89.84) b	85.4 (80.6)	99.7 (84.0)
	Multiplicity	3.97	3.18 b	1.94	3.25
	Refinement statistics
	Resolution	49.38 --2.25	49.04 --2.33	50.0 --2.77	42.62 --2.23
	Unique Refl.	55653(5436)	49514(4510)	25956(3884)	53417(3359)
	Rwork/Rfree (%)	18.2/24.3	20.8/25.8	30.0/34.6	17.26/22.64
	Total Atoms	9157	8895	8632	8707
	B--1.78	1.8450	1.49	1.81
	Ramachandran Plot
	Favored (%)	97	97	98.7	96
	Allowed (%)	3	2.73	1.2	3.73
	Disallowed (%)	0	0.27	0.1	0.27

a Values in parentheses are for the highest-resolution shell; b Rmerge = _hkl _i |Ii(hkl) -< I(hkl)>|/_hkl _i Ii(hkl).

Table S2

 S2 Characteristic parameters of YgjD, YgjD-YeaZ, YjeE and respective complexes calculated from the SAXS experimental curves. Analyses of the protein-ligand interaction by ITC. All the titrants and injectants except those in B are supplemented with 500 µM MgCl 2 . A, Titration of YgjD (30 µM) with AMPCPP (360 µM). B, Titration of the heterodimer YgjD-YeaZ (30 µM) with AMPCPP (360 µM). C, Titration of YjeE (57 µM) with ATPγS (680 µM). D, Titration of YjeE (50 µM) with ADP (600 µM). E, Titration of YjeE Y82A (28 µM) with ATPΥS (350 µM). F, Titration of YjeE W109A (41 µM) with ADP (492 µM). G, Titration of YgjD (30 µM) with ADP (360 µM). H, Titration of YeaZ (30 µM) with ADP (360 µM). I, Titration of YeaZ (30 µM) with ATP (360 µM). J, Titration of

					Quaternary	Rg Guin-	Rg P(r)	Dmax	MW	Theoretical	χ
					state	ier	(Å)	(Å)	(kDa)	a	MW(kDa)	b
	(Å) 32.0 E12A (30 µM) with AMPCPP (360 µM). K, Titration of YjeE YgjD-WT dimer 32.9 110 YgjD T43A (33 µM) with ADP (400 µM). 74.0 73.7 1.31 L, Titration of YjeE E108A (20 µM) with ADP (260 µM).
	YgjD	S97E			monomer	23.3	23.3	74	37.9	36.8	1.29
	YgjD-YeaZ			monomer	29.0	29.2	90	66.5	62.8	1.53
	YgjD-YeaZ-YjeE		monomer	29.3	29.0	90	81.3	80.7	1.42
	YgjD-YeaZ	1-219 -YjeE	monomer	28.9	28.8	89	75.9	78.5	1.38
	YgjD-YeaZ-YjeE	W109A	monomer	29.0	28.9	90	79.2	80.7	1.01
	YjeE				monomer	16.6	16.5	54	17.1	17.9	0.86
	YjeE				dimer	24.3	24.3	76	38.2	35.8	1.18
	YeaZ			dimer	26.3	26.4	84	51.4	52.0	1.50

a , Molar massM obtained from the whole I(q) curve (qmax=0.3Å) using the macromolecule volume and the SAXS-MoW method available at http://www.if.sc.usp.br/~saxs/. b , the theoretical masses were derived from the sequence taking into account the tags in each case. ▲ Fig.

S3

  The digested nucleosides of E. coli tRNA Lys alone. C. the digested nucleosides of E. coli tRNA Lys after enzymatic reaction with YgjD, YeaZ, YjeE and YrdC; the molecular weight of t 6 A was determined by MS (represented in the blue square). D. The digested nucleosides of E. coli tRNA Lys after enzymatic reaction with TCA and YgjD, YeaZ and YjeE. E. The digested nucleosides of E. coli tRNA Lys after incubation with YrdC. F. The digested nucleosides of E. coli tRNA Lys after incubation with YgjD, YeaZ and YrdC. G. The digested nucleosides of E. coli tRNA Lys after incubation with YjeE and YrdC. H. The digested nucleosides of E. coli tRNA

	Lys after enzymatic Lys S97E , YeaZ, YjeE and YrdC. I. The digested nucleosides of E. coli tRNA after enzymatic reaction with YgjD reaction with YgjD E12A , YeaZ, YjeE and YrdC. J. The digested nucleosides of E. coli tRNA Lys after enzymatic reaction with YgjD, YeaZ R118A , YjeE and YrdC. K. The digested nucleosides of E. coli tRNA Lys after enzymatic reaction with YgjD, YeaZ, YjeE E108A and YrdC. L. The digested nucleosides of E. coli tRNA Lys after enzymatic reaction with YgjD, YeaZ, YjeE W109A and YrdC.

MATERIALS & METHODS

Acknowledgement

The authors are grateful to M. Graille, L. Perrochia, T. Basta, P. Forterre, M. C. Daugeron for fruitful discussion throughout the experiment and manuscript.

Acknowledgment

We thank Andy Thompson (SOLEIL, St Aubin) for his assistance on the SAD data collection, Noureddine Lazar for his work on purification of the N 15labeled Gon7 and Ewen Lescop (ICSN, Gif s/Yvette for the NMR contribution.

Acknowledgement

We are grateful to Magali Aumont-Nicaise and Hélène Barreteau for their assistance in ITC and HPLC data collections, respectively. We thank Delphine Arquier for her assistance in LC/MS experiment. This work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. W. Zhang has been supported by a Ph. D fellowship from China Scholarship Council.

Supplementary materials

Structures of the Gon7/Pcc1 and Bud32/Cgi121 complexes provide insight into the t 6 A tRNA modification by yeast KEOPS/EKC

Supplementary figures

Fig. S1 The structural model of KEOPS from archaea [START_REF] Mao | Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine[END_REF]. The archaea KEOPS comprises Pcc1, Kae1, Bud32 and Cgi121, which are arranged in linear manner. Pcc1 functions as a dimerization module as indicated by an upward purple arrow.

Supplementary tables

Supplementary experimental

Primers in mutagenesis

The mutations and primers are listed in the table below. HPLC and MS analysis of the t 6 A 100 µl reaction mixture was applied to HPLC using a µRPC C2/C18 ST 4.6/100 column (pore size 120 Å) and nucleosides were detected by UV at 258 nm. The column was equilibrated with buffer A (10 mM Ammonium Acetate, pH 7.0). The gradient used for elution was 100% buffer A from 0 to 8 min followed by a linear gradient of 0-100% buffer B (6 mM Ammonium Acetate, pH 7.0 and 40% acetonitrile) from 8 to 30 min. The standard Cytosine (C), Uracil (U), Guanine (G), Adenine (A) (Sigma) and t 6 A (kindly provided by R.W.

Mutation

Adamiak, Polish Academy of Sciences) were used as reference for determination of the elution time of each digested nucleosides in the reaction mixture [START_REF] Adamiak | The chemical synthesis of the anticodon loop of an eukaryotic initiator tRNA containing the hypermodified nucleoside N6-/N-threonylcarbonyl/-adenosine/t6A/1[END_REF]. The area under each peak that corresponds to C, U, G, A and t 6 A was used for quantification of the digested nucleosides of tRNA.

The percentage of t 6 A is calculated as the average of area of t 6 A to area of C, U and G, respectively, normalized to tRNA alone and tRNA modified by wildtype YgjD, YeaZ, YjeE and YrdC. MS analyses of nucleosides were performed by mass spectrometer Micro TofQ (BRUKER DALTONICS) equipped with HPLC (Ultimate3000, DIONEX) using the µRPC C2/C18 ST 4.6/100 column. The gradient used in LC/MS was 100% buffer A from 0 to 8 min followed by a linear gradient of 0-100% buffer B (6 mM Ammonium Acetate, pH 7.0 and 40% acetonitrile) from 8 to 30 min. MS was employed to confirm the masses of C, U, G, A and t 6 A from digested nulceosides.

Supplementary tables

Table S1 Crystal data and structural refinement statistics.

Supplementary figures

Abstract

t 6 A (N 6 -threonylcarbamoyladenosine) is a modified nucleotide found in all tRNAs decoding ANN codons. Its role is to facilitate codon-anticodon pairing and to prevent frameshifting during protein synthesis. Universal distribution of t 6 A and its important role in fidelity of translation suggest that t 6 A was already present in the last universal cellular ancestor (LUCA). Recent genetic studies demonstrated that two universally conserved proteins of unknown function, Sua5 and Kae1 and their bacterial homologs YrdC and YgjD, are necessary for the synthesis of t 6 A in cells of Saccharomyces cerevisiae and Esherichia coli. In Bacteria the in vitro synthesis of t 6 A requires two other proteins specific for the bacterial domain. In Archaea and Eukaryoa, Kae1 is a subunit of a conserved heteromultimeric protein complex named KEOPS. Since its discovery in 2006, several studies have suggested involvement of KEOPS in diverse cellular processes in S. cerevisiae: transcriptional regulation, telomere homeostasis and genome maintenance. In this study we reconstituted the in vitro system for the synthesis of t 6 A modified tRNAs in archaeal and eukaryotic cells. We provide for the first time evidence that KEOPS from the hyperthermophilic archaean Pyrococcus abyssi and from S. cerevisiae together with Sua5 are necessary and sufficient for the catalysis of this reaction. We further demonstrated binding of both cognate and non-cognate tRNA to archaean KEOPS and detected two distinct ATP-dependent mechanisms of activation occurring in the course of the synthesis. Finally, we identified these activation mechanisms as being conserved in the three domains of life.

Received September 19, 2012;Revised November 9, 2012;Accepted November 11, 2012 Remarks 202

Remarks