Chapter Six -Strategies for crystallization of natural ribozymes

Benoit Masquida, D Sibrikova, Maria Costa

The group of ribozymes acting like turnover enzymes is up to now restricted to the ribonuclease P [START_REF] Guerrier-Takada | The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme[END_REF], the ribosome [START_REF] Cech | The Ribosome Is a Ribozyme[END_REF] and the spliceosome [START_REF] Galej | Molecular Mechanism and Evolution of Nuclear Pre-mRNA and Group II Intron Splicing: Insights from Cryo-Electron Microscopy Structures[END_REF]. Other natural ribozymes are endonucleolytic RNAs, catalyzing the reversible cleavage of a phosphodiester bond within their own chain. This group contains the hammerhead [START_REF] Hutchins | Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid[END_REF], hairpin [START_REF] Buzayan | Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA[END_REF], hepatitis delta virus (HdV) [START_REF] Sharmeen | Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage[END_REF], and the Varkud satelitte (VS) [START_REF] Saville | A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria[END_REF] ribozymes, and also group I [START_REF] Cech | In vitro splicing of the ribosomal RNA precursor of Tetrahymena: Involvement of a guanosine nucleotide in the excision of the intervening sequence[END_REF][START_REF] Michel | Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure[END_REF] and group II introns [START_REF] Michel | Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure[END_REF]. More recently discovered ribozymes include twister [START_REF] Roth | A widespread self-cleaving ribozyme class is revealed by bioinformatics[END_REF], twister sister, pistol and hatchet [START_REF] Weinberg | New classes of self-cleaving ribozymes revealed by comparative genomics analysis[END_REF], as well as GlmS [START_REF] Winkler | Control of gene expression by a natural metabolite-responsive ribozyme[END_REF], which is also a riboswitch.

When the cleavage intervenes at the boundaries of the ribozyme, like in the case of the HdV ribozyme, the structure of the initial, or pre-catalytic RNA is expected to be quite similar to the 3' cleavage product. Yet, most frequently, cleavage intervenes at an internal position. In the course of the reaction, the amount of pre-cleavage form decreases in solution until the catalytic equilibrium is reached. As fragments of the ribozyme itself, the RNA cleavage products may not be able to fold properly since cleavage affects the higher order structure of the RNA by splitting structural domains.

Therefore, a solution of active ribozymes ends up as a mix of different characteristic RNA precursors and products, to which should be added shorter species resulting from the degradation of unstable cleavage products.

Crystallization of biomolecules requires chemical and conformational homogeneity [START_REF] Kondo | RNA crystallization[END_REF].

RNA incubation in crystallization droplets usually lasts a couple of days or weeks at temperatures ranging from 15°C to 37°C, at pH comprised between 6 to 8, in the presence of monovalent and divalent and/or trivalent ions, like magnesium salts or coordinated transition metals such as cobalt, osmium or iridium that serve solving the phase problem using anomalous diffraction or scattering methods. Lanthanides can also be used for this purpose. Apart from crystallization agents such as ammonium sulphate, oily alcohols (MPD) or polyethylene glycols (PEG) of different molecular weights, various additives such as polyamines (spermine, spermidine) can be used. Even though not optimal, these conditions allow for some ribozyme activity, meaning that pre-and post-cleavage forms of the ribozyme may co-exist in the sample before the crystallization occurs. Preventing ribozymes to catalyze their own reaction is thus a mandatory aspect of ribozyme structural biology that requires designing unreactive constructs, yet still able to fold into the catalytically relevant conformation.

Endonucleolytic ribozymes catalyze RNA cleavage through a phosphodiester transesterification pathway based on the exchange of two phosphodiester bonds. The conservation of energy storage in this reaction allows for its reversibility. The reaction consists in a nucleophilic attack promoted by the 2'-hydroxyl group adjacent to the reactive phosphate group and generates 2',3'-cyclic phosphate and 5'-hydroxyl product termini. This 2'-OH group needs to be activated to become an efficient nucleophile to attack the 3'-adjacent phosphate group. However, the reaction relies on additional chemical and structural requirements. Going stepwise from activation to product formation, the catalytic pathway consists of successive chemical and conformational steps. Structural biologists need to find strategies to block the ribozymes under scrutiny at a single step in order to isolate conformers corresponding to a well-defined state along the catalytic pathway. As a general rule, success stems from good knowledge of the ribozyme catalytic behavior deduced from biochemical studies prior to the design of the RNA constructs, which will be assayed for crystallization. Several strategies can be employed to prevent the reaction to occur. This chapter reviews these strategies and illustrates them with representative examples of natural small ribozymes and selfsplicing introns.

Strategies to inactivate the nucleophile

Endonucleolytic ribozymes exhibit different chemical properties (exhaustively summarized in [START_REF] Lilley | How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes[END_REF]) to become efficient catalysts. In brief, the phosphodiester transesterification reaction consists in a bimolecular nucleophilic substitution (SN2).

The SN2-like reaction is characterized by the formation of the new bond, generating a pentavalent phosphorous intermediate, followed by cleavage of the leaving group, a mechanism that energetically culminates with the formation of a phosphorane transition state. This situation leads to chirality inversion if the atom targeted by the nucleophile is asymmetric. The transesterification mechanism assumes that the nucleophilic hydroxyl group is activated by deprotonation following action of a general base. The structure of the ribozyme provides an architecture that favors the placement of the nucleophile in line with the phosphorus and leaving 5'-oxygen atom. The alignment of these three atoms is required for the formation of the pentavalent phosphorane transition state, which adopts a trigonal bi-pyramidal geometry. The transition state needs also to be stabilized structurally by the ribozyme architecture, notably through an increased number of H-bonds. Finally, the leaving oxyanion should be stabilized by an acid, which gives a proton to stabilize the conjugated base, the 5'hydroxyl. This catalytic mechanism is very much similar to the one operated by the RNase A enzyme, in which all the above-mentioned atoms are aligned to perform the SN2-like reaction [START_REF] Raines | Ribonuclease A[END_REF]. All these steps add on individually to result in the final catalytic rate. Vice-versa, disturbance of any of these steps decreases the overall reaction rate. In principle, the modification of the chemical groups involved in any of these events can help decrease the overall catalytic rate sufficiently to render the ribozyme inactive enough over the incubation time necessary for crystallization. The most straightforward strategy is to remove or replace the nucleophilic oxygen atom by molecular engineering.

Another strategy consists in mutating the residues that will either activate the nucleophile or stabilize the leaving group. Finally, the scissile phosphate group can be omitted by synthesis of split oligonucleotides or by circular permutation.

Another critical aspect regarding ribozymes crystallization is related to the integrity of their architecture. The 5'-and 3'-boundaries of ribozymes are not always easy to define. Moreover, loops can contain additional domains dispensable for the ribozyme catalytic architecture. Usually, efforts are made to reduce the length of the ribozyme constructs while retaining significant catalytic activity. This strategy can lead to the removal of regions that are somewhat dispensable for the catalysis but critical for the architecture .

Nevertheless, some domains may also confer to the ribozymes too much flexibility, which may prevent folding as a single conformation. In these cases, adequate domain replacement monitored by conformational analysis can help impr ove the situation.

Illustrations of these situations will be given in the paragraphs below.

When the cleavage site is at the edge of the ribozyme

The location of the cleavage site at the 5'-boundary of the ribozyme constitutes the most ideal situation, since the 3'-RNA product corresponds to the entire ribozyme domain and the other one to a dispensable short fragment. The cases of the hepatitis delta (HdV, [START_REF] Ferré-D'amaré | Crystal structure of a hepatitis delta virus ribozyme[END_REF]) and of the GlmS [START_REF] Klein | Structural basis of glmS ribozyme activation by glucosamine-6-phosphate[END_REF][START_REF] Cochrane | Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor[END_REF] ribozymes well illustrate this situation, where the ribozyme domains are located downstream from the cleavage site. The cleavage product corresponding to the ribozyme core can be purified from an in vitro cleavage assay. Yet, the study of the catalytic mechanism requires the presence of the scissile nucleotide. The latter can be added by hybridization of a ribozyme domain made from an oligonucleotide mimicking the unreacted substrate by bearing a specific chemical modification inhibiting catalysis.

When the structure of the HdV ribozyme [START_REF] Ferré-D'amaré | Crystal structure of a hepatitis delta virus ribozyme[END_REF][START_REF] Ferre-D'amare | Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module[END_REF] was solved (Figure 6.2.1), the only RNA structures that were known apart from a variety of helical structures, were of tRNAs [START_REF] Sussman | Crystal structure of yeast phenylalanine tRNA. I. Crystallographic refinement[END_REF][START_REF] Westhof | Crystallographic refinement of yeast aspartic acid transfer RNA[END_REF], of two versions of the hammerhead ribozymes [START_REF] Pley | Three-dimensional structure of a hammerhead ribozyme[END_REF][START_REF] Scott | The crystal structure of an all-RNA hammerhead ribozyme : A proposed mechanism for RNA catalytic cleavage[END_REF], and of the P4-P6 domain from the Tetrahymena intron [START_REF] Cate | Crystal structure of a group I ribozyme domain: principles of RNA packing[END_REF]. In this context, the main goal was to expand the RNA structural repertoire by solving the structure of a new RNA architecture. When embedded in a viroid, the ribozyme allows to cleave the multimeric RNA copy resulting from the viroid RNA replication through a rolling circle mechanism into individual genomes and simultaneously to ligate their ends to form circles [START_REF] Branch | A replication cycle for viroids and other small infectious RNA's[END_REF]. In spite of the absence of sequence requirements and of need for catalytic ions, the 5'-hydroxyl cleavage product of the ribozyme was purified to sort out the misfolded intermediates and meet the chemical and conformational homogeneity of the sample required for crystallization. Among the structural features unraveled by the first crystal structure of the HdV ribozyme [START_REF] Ferré-D'amaré | Crystal structure of a hepatitis delta virus ribozyme[END_REF], a pseudoknot (P1.1) formed by two consecutive G=C base pairs was observed between residues from loop L3 and the junction of stems P1 and P4 (J1/4), in addition to the one already known from comparative sequence analysis that leads to the formation of the P2 stem. P1.1 is actually instrumental for positioning the first residue of the ribozyme 3'-cleavage product, G+1, which is involved in a wobble pair with U37. Residue C75 has been identified as the residue responsible for the limiting proton transfer step of the catalytic reaction. C75 should either abstract the proton of the 2'-hydroxyl of U-1 [START_REF] Tanner | Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes[END_REF][START_REF] Shih | Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage[END_REF] or stabilize the 5'-oxyanion of the leaving group [START_REF] Nakano | Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism[END_REF]. In agreement with these data, the crystal structure shows that the N3 Watson-Crick group of C75 establishes a hydrogen bond with the 5'-hydroxyl group of G1. However, this property would imply that the pKa of C75(N3) were was around ~7 instead of 4.2 as observed for the free cytosine. This hypothesis was addressed by NMR, and only a weak increase of the pKa was observed in the context of the HdV cleavage product [START_REF] Luptak | Direct pK(a) measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme[END_REF].

Further crystallographic studies were carried out to investigate the structure of the precleavage form of the ribozyme. Catalysis was prevented in these constructs following strategies that will be presented below.

The GlmS RNA is a ribozyme and also a riboswitch that binds glucosamine -6-phosphate (GlcN6P), a bacterial and fungal cell wall precursor [START_REF] Milewski | Glucosamine-6-phosphate synthase--the multi-facets enzyme[END_REF]. The cellular concentration of GlcN6P negatively controls the RNA amount of glms, the gene producing the GlcN6P synthase [START_REF] Winkler | Control of gene expression by a natural metabolite-responsive ribozyme[END_REF]. While several crystal structures of the aptamer domain of this riboswitch have been solved, the folding energetic landscape between the GlcN6P bound and unbound conformations of the riboswitch have only been investigated recently [START_REF] Savinov | Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element[END_REF]. The first structural study from the Thermoanaerobacter tengcongensis GlmS constructs gathered crystal structures with or without the scissile residue allowing comparison between the conformations of the pre-and post-cleavage forms [START_REF] Klein | Structural basis of glmS ribozyme activation by glucosamine-6-phosphate[END_REF][START_REF] Klein | Crystallization of the glmS ribozymeriboswitch[END_REF]. Yet, the binding of glucose-6-phosphate (Glc6P), an inhibitor of the GlmS riboswitch, but not of GlcN6P could be observed. Only models of the catalytic mechanism could be deduced from these structures. Just after reporting the crystal structure of the T. tengcongensis GlmsS riboswitch, another research group published the crystal structure of the Bacillus anthracis GlmS riboswitch in the presence of the ligand (Figure 6.2.2b). The direct involvement of the amino group of GlcN6P to protonate the leaving 5'-oxyanion during catalysis could be demonstrated [START_REF] Cochrane | Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor[END_REF]. This set of crystal structures shows that when cleavage intervenes at the 5'-end, the addition of a couple of 5'-nucleotides does not have major effects on the overall ribozyme architecture. However the addition of 5'-nucleotides restores the cleavage site, requiring inhibition of the nucleophile activation. Strategies used to prevent nucleophile activation are described below.

Removal or neutralization of the catalytic 2'-hydroxyl group

With a lesser knowledge of the catalytic mechanism, meaning when the nucleophile position has been identified, but not much other informations are available, the easiest strategy is either to remove the 2'-hydroxyl group or to modify it by methylation.

Removal may lead to a loss of information by releasing some of the constraints on the active nucleotide, while methylation preserves the 2'-O-atom, which potentialy confers the opportunity to observe its role in catalysis.

The hammerhead ribozyme

The hammerhead ribozyme was the first catalytic RNA, the structure of which was solved by crystallography [START_REF] Pley | Three-dimensional structure of a hammerhead ribozyme[END_REF]. The ~50 nt hammerhead ribozyme was found in the 80's in the satellite RNA of the avocado sunblotch viroid [START_REF] Hutchins | Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid[END_REF]. More recently, genome surveys have revealed that the hammerhead motif is actually widespread in genomes, from bacteria to humans [START_REF] Jimenez | Structure-based search reveals hammerhead ribozymes in the human microbiome[END_REF]. This fairly short RNA allowed for defining a trans-acting construct based on the hybridization of a ribozyme domain and a substrate strand [START_REF] Fedor | Substrate sequence effects on "hammerhead" RNA catalytic efficiency[END_REF][START_REF] Ruffner | Studies on the hammerhead RNA self-cleaving domain[END_REF] that acts like a true enzyme/substrate complex.

In the first hammerhead crystal structure, a minimal ribozyme domain was produced by in vitro transcription, whereas the substrate strand was chemically synthesized as a DNA strand. This strategy allowed for the systematic replacement of all 2'-hydroxyl groups of the substrate strand by hydrogen atoms and consequently, prevented cleavage at the scissile phosphate. Since the RNA strand geometry cannot fold like B-DNA due to the presence of the 2'-hydroxyl groups, the DNA strand was forced to adopt the A-form [START_REF] Pley | Three-dimensional structure of a hammerhead ribozyme[END_REF]. In the structure of the all-RNA hammerhead ribozyme published shortly afterward, a two strands strategy to reconstitute the minimal ribozyme was again chosen. In this case, the nucleotide bearing the proton of the nucleophilic 2'-hydroxyl group was changed to a 2'-O-methyl group to block catalysis [START_REF] Scott | The crystal structure of an all-RNA hammerhead ribozyme : A proposed mechanism for RNA catalytic cleavage[END_REF]. In order to impair catalysis during crystallization, neutralization of the nucleophile by chemical modification is preferred over the depletion of the attacking 2'-hydroxyl group. This is because the former strategy is more likely to provide relevant insight into the catalytic mechanism.

However, introduction of such chemical modifications relies on solid-phase chemical synthesis (See [START_REF] Caruthers | A brief review of DNA and RNA chemical synthesis[END_REF][START_REF] Hartsel | RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry[END_REF] for Review) of short RNA oligonucleotides carrying the desired modified nucleotides. Indeed, the all-RNA hammerhead ribozyme showed the same conformation as the DNA-RNA hybrid construct. Yet, in both crystal structures, the conformation of the active site was not in agreement with the biochemical and enzymatic data previously obtained for the ribozyme. Also, in these structures, the conformation at the cleavage site did not show the expected alignment of the three atoms involved in the transesterification reaction (Figure 6.2.3a), namely the 2'hydroxyl of the catalytic residue on one side, and the phosphorus atom and the 5'-hydroxyl groups of the contiguous 3'-residue on the other side. Subsequently, intensive research was conducted using the minimal RNA construct to try to observe its active conformation by freeze-trapping an active version of the ribozyme at low pH in the presence of magnesium ions [START_REF] Scott | Capturing the structure of a catalytic RNA intermediate: The hammerhead ribozyme[END_REF]. But again, the conformation trapped in the crystal was still not fully supportive of an in-line SN2-like mechanism for the self-cleavage reaction.

The answer actually came 10 years later when the crystal structure of a longer hammerhead ribozyme derived from Schistosoma mansoni was solved (Figure 6.2.3b).

This hammerhead ribozyme version comprises additional critical tertiary contacts between peripheral segments that improve catalysis by 1000-fold at sub-millimolar magnesium concentration [START_REF] Khvorova | Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity[END_REF][START_REF] Canny | Fast cleavage kinetics of a natural hammerhead ribozyme[END_REF]. For crystallization purposes, a two-strand construct was also used to reconstitute the ribozyme and the nucleophilic 2'-hydroxyl group was again neutralized by methylation. The resulting crystal structure revealed that the active site of this longer ribozyme forms a 3-way junction. Stem II is closed by an apical loop, which interacts with the internal loop embedded in stem I, itself formed between the ribozyme and the substrate strand [START_REF] Martick | Tertiary contacts distant from the active site prime a ribozyme for catalysis[END_REF]. This peripheral tertiary interaction induces a major conformational change in the distal 3-way junction that forces the in-line positioning of the tree atomic groups involved in the catalytic reaction. The right panel of Figure 6.2.3 shows the very different positions adopted by the dinucleotide around the scissile bond. Taken together, these observations indicate that the historical hammerhead construct that was used as a model system for a long time despite its poor activity had in fact been too much shortened to keep the relevant pre-catalytic conformation [START_REF] Nelson | When to believe what you see[END_REF]. In conclusion, designing a streamline version of a ribozyme for crystallization purposes should rely, first on careful sequence analysis of the available ribozyme variants and, second on careful biochemical and kinetic analysis of each construct in order to select the molecule that more closely mimics the natural full-length version.

Group I intron

The ~300 nt group I ribozymes are able to self-splice along a two-step transesterification mechanism. First, an exogenic guanosine (exoG) binds a pocket in the catalytic core to play the role of the nucleophile. During the first step, exoG transesterifies the bond between the 5'-exon and the intron and remains attached to the 5'-end of the intron. Following a profound conformational change leading to the replacement of exoG by the ultimate G residue (G) of the intron in its pocket, the 3'residue of the 5'-exon cleaves the bond between the intron and the 3'-exon (Figure 6.2.4a). This pathway results in exon ligation and intron release. Capturing intermediates along this complex pathway requires inactivation of well-chosen residues (Figure 6.2.4b, c).

The self-splicing group I intron from the pre-tRNA Ile of bacterium Azoarcus [START_REF] Adams | Crystal structure of a self-splicing group I intron with both exons[END_REF][START_REF] Adams | Crystal structure of a group I intron splicing intermediate[END_REF], was crystallized bound to its exons in a conformation preceding the second catalytic step of splicing. This intron/exons complex was stabilized by four specific 2' -deoxy substitutions: two at intron positions A205 and ωG, one at the end of the 5'-exon and a fourth one at the beginning of the 3'-exon. Collectively, these modifications slow down exon ligation by a million-fold and allow for crystal nucleation and growth over ~2 weeks. The crystallization construct was obtained by hybridization of a transcript corresponding to the majority of the intron with an RNA-DNA oligonucleotide spanning the 3'-section of the intron and the beginning of the 3' -exon, and a trinucleotide mimicking a short 5'-exon ending with a dT residue. This three-piece strategy greatly facilitated introduction of the substitutions since all of the 2' -deoxy nucleotides were inserted during chemical synthesis of the RNA-DNA chimeric oligonucleotides (Figure 6.2.4). In addition, a U1A binding site was introduced in the L6 loop of the intron to optimize crystallization by promoting crystal packing interactions between molecules in solution [START_REF] Ferre-D'amare | Use of the spliceosomal protein U1A to facilitate crystallization and structure determination of complex RNAs[END_REF]. Note that U1A contains Seleno-methionine residues used to solve the phase problem without performing heavy atom soaking or co -crystallization.

The crystallized intron/exons complex revealed the structural basis for 5' -and 3'-splice site selection and substrate alignment used by group I intron ribozymes. However, positioning of the metal ions into the active site was found not to be fully consistent with available biochemical data presumably because of the 2'-deoxy substitution at the terminal intron guanosine ωG [START_REF] Adams | Crystal structure of a self-splicing group I intron with both exons[END_REF]. Indeed, only subsequent crystallization of an allribose version of the same construct, containing the catalytically essential ωG O2'-ligand, allowed the correct positioning of the metal ions in the active site and provided crystallographic evidence for the 'two-metal-ion' mechanism of group I intron splicing [START_REF] Stahley | Structural evidence for a two -metal-ion mechanism of group I intron splicing[END_REF]. In this case the same 5'-exon (CAT) trimer was used to reduce sufficiently the exon ligation reaction and permit crystal nucleation. Again, this example nicely illustrates that crystallization of complete ribozymes in their active state is a challenging task: modification of catalytically important positions is necessary to block or reduce the activity of the ribozyme, still, such changes should not alter the catalytically competent structure of the active site.

Other structures of group I introns have been solved by X-ray crystallography [START_REF] Guo | Structure of the tetrahymena ribozyme; base triple sandwich and metal ion at the active site[END_REF][START_REF] Golden | Crystal structure of a phage Twort group I ribozyme-product complex[END_REF].

Yet, their conformations did not represent any active state, and thus did not require introduction of any chemical modification. In the structure of the Tetrahymena ribozyme [START_REF] Guo | Structure of the tetrahymena ribozyme; base triple sandwich and metal ion at the active site[END_REF], the substrate helix was not included in the construct. In the ribozyme of the orf142 from the phage Twort [START_REF] Golden | Crystal structure of a phage Twort group I ribozyme-product complex[END_REF], the ribozyme core was crystallized with a tetrameric RNA mimicking the substrate. However, since this pseudo-substrate does not correspond to any catalytic step, proper docking onto the ribozyme core could not be observed.

Group II intron

Group II introns, which are not evolutionarily related to group I introns, constitute the second class of self-splicing ribozymes known to date. They are abundant in bacteria and bacterial-derived organelles of fungi, plants and algae. Group II introns are large catalytic RNAs of ~600 nucleotides which, in association with the reverse transcriptase they encode, can also behave as mobile retrotransposable elements. Despite their bacterial origin, it is now widely believed that group II introns played a determinant part in eukaryotic evolution as the ancestors of the nuclear premessenger introns and their splicing machinery (the spliceosome) [START_REF] Lambowitz | Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution[END_REF]. This evolutionary hypothesis is grounded on the extensive structural and mechanistic similarities shared by the two splicing systems.

Both group II intron and nuclear pre-mRNA splicing proceed through two sequential transesterification reactions and generate a typical intron form called the 'lariat' intron.

Self-splicing of group II introns is initiated by the 2'-hydroxyl group of a highly conserved adenosine, lying unpaired in domain VI (bpA in Figure 6. Recent X-ray crystal structures of an excised group II intron lariat, alone or bound to its 5'-exon substrate (Figure 6.2.5), uncovered the structural basis for reverse splicing by group II introns [START_REF] Costa | Crystal structures of a group II intron lariat primed for reverse splicing[END_REF]. These structures revealed for the first time that the 2'(A)-5'(G) branch plays a direct and crucial role in organizing the active site for the second step of splicing. Remarkably, the structures also show that after completion of the splicing reaction, the last nucleotide of the freed intron lariat remains firmly positioned in the catalytic center with its 3'-OH activated by a highly coordinated metal ion (M1 in Figure 6.2.5c) and poised for catalysis of the reverse splicing reaction. In the absence of its 5'and 3'-exon substrates, the excised group II lariat is a stable RNA, therefore, the purified lariat sample could be crystallized without any modification. Formation of a stable lariat/5'-exon complex, however, required co-crystallization of the lariat with an unreactive analog of the 5'-exon. This analog consisted of a 7-mer RNA carrying a 3'deoxy ribose at its 3'-end (Figure 6.2.5a, c). Removal of the terminal 3'-hydroxyl group of the 5'-exon was necessary to block lariat 'debranching': a reaction that is mechanistically analogous to the reverse of the first step of splicing and, accordingly, leads to the production of linear intron molecules covalently attached to the 5'-exon. Comparison of the lariat structures with and without this substrate revealed that binding of the 5' -exon drives a local conformational rearrangement of domain V that extends into the active site and contributes to coordination of a second catalytic metal ion. Interestingly, native crystals (grown in magnesium-containing solutions) lacked electron density corresponding to this second metal ion, most probably due to the absence of the critical 3'-OH group of the 5'-exon. Evidence for this second catalytic metal ion was nevertheless obtained through crystal soaking in ytterbium -an anomalous scatterer lanthanide that mimics highly coordinated magnesium ions, but binds with higher affinity. Indeed, ytterbium soakings revealed the presence of two metal ions in the reaction center (Y1 and Y2 in Figure 6.2.5c), one of which perfectly superimposes with metal ion M1 already identified in the native crystals. Altogether, the two sets of crystals allowed the direct observation of all but three metal ion coordinations that build-up the 'two-metal ion' transition state for the reverse splicing reaction (Figure 6.2.5c; [START_REF] Costa | Crystal structures of a group II intron lariat primed for reverse splicing[END_REF]). Importantly, the transition state model derived from this crystallographic work is identical to the one prevailing for group I intron self-splicing (see previous section; [START_REF] Stahley | Structural evidence for a two -metal-ion mechanism of group I intron splicing[END_REF]).

The hairpin ribozyme

The hairpin ribozyme provides another example where the precise definition of the domains important for folding was critical to solve a catalytically relevant structure.

Like the hammerhead and HdV ribozymes, the hairpin ribozyme is part of the satellite RNA of a virus (tobacco ringspot virus), and chops the multimeric RNA resulting from a rolling circle amplification [START_REF] Buzayan | Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA[END_REF]. The hairpin ribozyme was named after the shape of the secondary structure of the minimal fragment initially studied (Figure 6.2.6). This construct consists in two helical domains (A and B), each incorporating an internal loop [START_REF] Hampel | Hairpin' catalytic RNA model : evidence for helices and sequence requirement for substrate RNA[END_REF]. In the initially characterized hairpin ribozyme construct, a kink between domains A and B promotes docking of the internal loops so that catalysis occurs. However , structural studies in solution have shown that the linker between the domains is very flexible [START_REF] Murchie | Folding of the hairpin in its natural conformation achieves close physical proximity of the loops[END_REF][START_REF] Walter | Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction[END_REF]. Closer analysis of the natural structural context indicated that the ribozyme actually folded from a four way junction, bringing into play two additional helices that stack on top of domains A and B in order to form an RNA holiday-like junction [START_REF] Mckinney | Single-molecule studies of DNA and RNA four-way junctions[END_REF]. These junctions, like the related DNA holiday junctions instrumental in genomic recombination events [START_REF] Lilley | The Holliday junction is finally seen with crystal clarity[END_REF], form an "X" with each branch corresponding to an helix.

The 5'-helices of domains A and B are tethered so that a kink between them results in docking of internal loops A and B. In a minimal construct presenting only domains A and B, this situation results in high conformational heterogeneity due to constant competition between helical stacking at the edge of the two domains and docking of the internal loops. FRET experiments indicated that upon folding, the natural 4-way junction was forming a dominant conformation forcing the two domains required for catalysis to dock productively. The 4-way junction context thus increases dramatically the rate of interaction between domains A and B as compared to the original and much more flexible hairpin construct corresponding to the minimal construct [START_REF] Murchie | Folding of the hairpin in its natural conformation achieves close physical proximity of the loops[END_REF]. This 4-way junction hairpin ribozyme RNA to which a U1A protein binding site was added to improve crystallization [START_REF] Ferre-D'amare | Use of the spliceosomal protein U1A to facilitate crystallization and structure determination of complex RNAs[END_REF] was finally solved by X-ray crystallography [START_REF] Rupert | Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis[END_REF]. Like in the case of the all-RNA hammerhead ribozyme, the nucleophilic 2'-hydroxyl group from the substrate strand was neutralized by methylation. The structure showed close to perfect alignment between the three atoms involved in the SN2-like reaction as well as pointed to a network of hydrogen bonds stabilizing the pre-catalytic state. In particular, the scissile G residue is pulled out from domain A by forming a trans Watson-Crick base pair with an unpaired C of domain B, which orients its backbone favorably for the S N2-like reaction (Figure 6.2.6d). It is worth to note that the structure of an all-RNA substrate hairpin ribozyme could be solved despite its catalytic activity. In this case, the constraints from the crystal packing together with the occurrence of the ligation reaction seem to prevent product strands dissociation [START_REF] Rupert | Transition state stabilization by a catalytic RNA[END_REF].

In order to go beyond the structure of the ground state and to elucidate the catalytic mechanism, another modification strategy of the ribozyme was undertaken to study the structure of the transition state. This strategy relies on the coordination of both the 2'and 3'-hydroxyl groups of the residue bearing the nucleophile and of the 5'-hydroxyl group of the downstream nucleotide by a vanadate ion. Vanadate coordination fairly well mimics the phosphorane intermediate generated during the cleavage reaction [START_REF] Rupert | Transition state stabilization by a catalytic RNA[END_REF].

To allow binding of the vanadate ion, the substrate was split into two fragments resembling the reaction products with free 2'-, 3'-and 5'-hydroxyl groups. This strategy leads to the removal of the scissile phosphate, which is replaced by the vanadate ion.

The crystal structure reveals how the transition state is stabilized throughout increasing the number of hydrogen bonds formed with the two nucleotides in the vicinity of the vanadate (Figure 6.2.6e). Noteworthy, the structure of the ribozyme with a split substrate displaying a 2',3'-cyclic phosphate at the cleavage site could also be solved to complete the snapshots necessary to describe the entire catalytic process [START_REF] Rupert | Transition state stabilization by a catalytic RNA[END_REF]. The structure of the transition state of the hammerhead ribozyme was also studied using vanadate ion binding to the cleavage site [START_REF] Mir | Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction[END_REF][START_REF] Mir | Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue[END_REF]. The catalytic site undertakes a substantial reshaping under these conditions and allows for describing more accurately the role of the residues and ions potentially involved in the reaction, notably G12, the general base, and the two magnesium ions close to the scissile bond. The vanadate structure also suggests that a water molecule bound to a divalent cation could act as the general acid that stabilizes the 5' oxy-anion.

Removal of the scissile phosphodiester bond using circular permutation

Success of the split substrate strategy relies on efficient hybridization of the RNA strands to their target as in the case of the hairpin ribozyme. However, this pre-requisite is not always met since annealing efficiency depends on the length and concentration of three different RNA species, each one potentially engaged in individual folding events that might perturb the formation of a well-folded trimer. Moreover, if the substrate binds to a region with an intricate fold, hybridization itself may be inefficient. An example that illustrates this situation is given with the lariat capping ribozyme (LC) [START_REF] Meyer | Speciation of a group I intron into a lariat capping ribozyme[END_REF], which is the topic of an entire chapter in the present book (Chapter X). In brief, this ribozyme catalyzes a transesterification reaction called 'branching' that is initiated by the nucleophilic attack of a 2'-hydroxyl group from a U residue on a C residue residing two nucleotides upstream. This reaction leads to the formation of a short 3 nt lariat in which the U residue is covalently attached to the C residue through a 2',5'phosphodiester bond [START_REF] Nielsen | An mRNA is capped by a 2', 5' lariat catalyzed by a group I-like ribozyme[END_REF]. From a chemical point of view, the lariat is identical to those generated by group II introns and the spliceosome during the first step of splicing [START_REF] Galej | Molecular Mechanism and Evolution of Nuclear Pre-mRNA and Group II Intron Splicing: Insights from Cryo-Electron Microscopy Structures[END_REF], except that the lariat loops involved in splicing span hundreds of nucleotides instead. In the case of the LC ribozyme, the 2',5'-branch point structure is well buried into the ribozyme core, where each nucleotide from the lariat weaves specific stacking and hydrogen bond interactions with specific core residues. These non-canonical interactions could not be deduced from comparative sequence analysis since the lariat sequence is fully conserved among all LC ribozyme homologues [START_REF] Tang | Molecular characterization of a new member of the lariat capping twin-ribozyme introns[END_REF]. Most importantly, the sequence stretch 3' from the cleavage site directly contributes building up the 3-way junction of the regulatory domain, which works by forming a receptor for a residue within a loop of the ribozyme core, instrumental for structuring the catalytic site. Initial efforts to crystallize the ribozyme using synthetic oligonucleotides were unsuccessful, because the hybrids could not properly fold.

To overcome this problem, a circular permutation strategy was implemented to swap the extremities of the ribozyme to the residues around the scissile phosphodiester , while the natural ends located in the stem were tethered by a UUCG tetraloop [START_REF] Meyer | Cis-Acting 5' Hammerhead Ribozyme Optimization for In Vitro Transcription of Highly Structured RNAs[END_REF] (Figure 6.2.7). Without any starting G residues, the 5'-CAU sequence resulting from this permutation did not follow at all the sequence requirements of the enzyme used to produce the RNA in vitro, the T7 RNA polymerase. For production purpose, a 5'hammerhead ribozyme domain was thus inserted upstream from the LC ribozyme. Since the hammerhead was introduced to increase the transcription yield of the LC ribo zyme, the optimal sequence transcribed by the T7 RNA polymerase was introduced right downstream from the T7 promotor [START_REF] Meyer | Cis-Acting 5' Hammerhead Ribozyme Optimization for In Vitro Transcription of Highly Structured RNAs[END_REF]. The version of the ribozyme based solely on the 3-way junction proved to be inefficient at cleaving, certainly because the LC ribozyme could fold faster, preventing the hammerhead ribozyme to adopt a structure prone to cleave. Consequently, the hammerhead was optimized for fast-folding by preserving the tertiary contacts observed in the Schistosoma ribozyme [START_REF] Canny | Fast cleavage kinetics of a natural hammerhead ribozyme[END_REF]. In addition, an HdV ribozyme was added at the 3'-end of the LC ribozyme to guarantee that the construct would not present any additional residue after its terminal G, G.

The structure of the circularly permutated LC ribozyme allowed to uncover all the interactions that the three lariat residues mediate with the other nucleotides from the core. It is worth noting that a water molecule interacts with the 2'-hydroxyl of U232 and the 5' hydroxyl of C230, and could be considered reminiscent of the scissile phosphate group that is available in the natural LC ribozyme, but missing in the circularly permutated form due to the construct design (Figure 6.2.7). Nevertheless G, which is important for catalytic activity, could not be observed due to structural disorder. This indicates that the conformation captured in the crystal corresponds to a snapshot of the LC ribozyme right after catalysis and before release of the RNA products.

Interestingly, circular permutations intervene naturally in RNA sequences. Domain shuffling can lead to RNA circular permutations, notably in back-splicing events resulting in the appearance of circular RNAs originating from pre-mRNAs [START_REF] Li | The Biogenesis, Functions, and Challenges of Circular RNAs[END_REF][START_REF] Franz | Circular RNAs: a new class of biomarkers as a rising interest in laboratory medicine[END_REF]. As far as the active RNA structure is maintained, the position of the 5'-and 3'-ends can be located in different regions. Along the same idea, loops closing helices can adopt any length from 3 nt to several kb as far as the few nucleotides contributing to tertiary interactions are preserved, if any. Such examples of natural circularly permutated RNAs have been described for the signal recognition particle (SRP) [START_REF] Plagens | Circularization restores signal recognition particle RNA functionality in Thermoproteus[END_REF] and for the hammerhead and HdV-like ribozymes [START_REF] Hammann | The ubiquitous hammerhead ribozyme[END_REF][START_REF] Webb | HDV-like self-cleaving ribozymes[END_REF]. Circular permutations can also be engineered to incorporate chemically modified residues further used in biochemical studies [START_REF] Gott | Using circular permutation analysis to redefine the R17 coat protein binding site[END_REF][START_REF] Harris | Use of circular permutatio n and end modification to position photoaffinity probes for analysis of RNA structure[END_REF][START_REF] Ohuchi | Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation[END_REF][START_REF] Truong | Designing fluorescent biosensors using circular permutations of riboswitches[END_REF]. Examples involving ribozymes include the RNase P where circular permutations were engineered to insert photo-agents to perform cross-linking studies [START_REF] Harris | Use of circular permutatio n and end modification to position photoaffinity probes for analysis of RNA structure[END_REF] or to study folding kinetics [START_REF] Pan | Pathway modulation, circular permutation and rapid RNA folding under kinetic control[END_REF].

In principle, circular permutation (CP) can be engineered on any RNA, provided that the 5'-sequence follows the requirement of the RNA polymerase that will be used to transcribe it from the DNA template. CP is thus a method of choice to express an RNA as a single molecule, based on the idea that the removal of the scissile phosphate group does not prevent homogenous folding.

Mutation of residues involved in the acido-basic aspects of transesterification

The 154 nt Varkud Satellite (VS) ribozyme is a fairly large ribozyme within the family of nucleolytic ribozymes. It is found in the mitochondria of a specific isolate of the Neurospora fungus [START_REF] Saville | A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria[END_REF]. Inactivation of the VS ribozyme for crystallization purpose was achieved following an original strategy based on modifying the residues involved in the proton transfer necessary for stabilizing the catalytic intermediates [START_REF] Suslov | Crystal structure of the Varkud satellite ribozyme[END_REF]. In brief, its structure is organized around three 3-way junctions encompassing seven helices (numbered 1 to 7). From each of those 3-way junctions protrudes one stem-loop, each one contacting a specific region of the ribozyme (Figure 6.2.8). The stacking between stems from the 3-way junctions generates a helical region spanning four helices (1 to 4).

The structure shows a feature so far unique to this ribozyme. The ribozyme crystallizes as a dimer in which the substrate stem (numbered 1) of one ribozyme docks in the second ribozyme in a catalytically active conformation [START_REF] Suslov | Crystal structure of the Varkud satellite ribozyme[END_REF]. This conformation is characterized by the formation of a kissing complex between the loops of helices 1 from one ribozyme and the loop from helix 5 of the second. Interestingly, these two 7 nt loops adopt similar conformations reminiscent of the anticodon loop of tRNAs. In each loop, a U-turn intervenes after the second nucleotide allowing the next four ones to adopt a conformation consistent with the formation of a four base pairs helix. In tRNAs, the fourth helical nucleotide is often hyper-modified to avoid decoding mistakes [START_REF] Fernandez-Millan | Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology[END_REF]. The trans interaction between loops 1 and 5 leads to the docking of the substrate along helices 2 and places the internal loop from helix 6 in position to build up the active site.

The observed dimer is in agreement with former biochemical, NMR data, and SAXS structure determination [START_REF] Wilson | Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid-base catalysis? A critical assessment of available experimental data[END_REF].

The strategy implemented by the authors was motivated by obtaining the RNA as a single strand to be able to purify it under native conditions since the dimer formed in the course of the transcription reaction. The structures of two mutants were solved, each one strongly affecting proton transfer in the catalytic site. The G638A structure was solved by single wavelength anomalous diffraction (SAD), and the A756G structure was solved by the molecular replacement method using the G638A structure as a search model. Residues important for catalysis locate in stems 1 and 6. In helix 1, G638 acts as a general base to abstract the proton of the 2'-hydroxyl group of the cross-strand stacked guanine. Helix 6 harbors A756, the general acid that stabilizes the leaving oxy-anion after the formation of the phosphorane transition state. The two X-ray structure models of the mutant forms G638A and A756G of the VS ribozyme unravel how the two residues contribute both to the structure and to the catalysis.

In the G638A structure, the O2', phosphorus and O5' atoms of the scissile bond between G620 and A621 are not perfectly in line, showing that the mutation significantly affects the activation of the reaction (Figure 6.2.8c). The extrusion of A621 from its helix and further stabilization through A-minor binding to a G=C pair in helix 2 indicate how the in-line positioning of the three atoms taking part in the SN2-like reaction may be easily achieved. However, A638, which stacks on the face of G620 points its Watson-Crick edge towards the phosphate group of A621. This situation places N1 of A638 in close enough proximity of the O2'-group from G620 and supports the notion that G638 is the general base involved in activation of the reaction. In the G638A crystal structure, the general acid A756 also points towards the phosphate group of A621. Noteworthy, a water molecule is clamped by hydrogen bonds between N1 of A756 and O2P of A621. The position of this water molecule could mimic the O5'-ligand of the phosphorane intermediate that would be resolved by proton transfer from A756, which would be protonated on its N1 atom (Figure 6.2.8).

In the A756G structure model (Figure 6.2.8d), the three groups involved in the SN2-like reaction are placed in-line much better than in the G638A VS ribozyme RNA. Similar to the G638A crystal structure, A621 is extruded from its helix to make an A-minor contact within the shallow groove of helix 2. The distance between the O2'-nucleophile and the N1 group of G638 is increased by the C3'-endo pucker of the G620 ribose. This group would actually reside in a closer distance, if the ribose were in a C2'-endo conformation.

The offset of the O2' leaves the N1-and N2-groups of G638 free to form hydrogen bonds with the O1P and O2P atoms of A621. The O2P-and O5'-groups of A621 are also in contact with the N1-and N2-groups from G756. All these contacts are consistent with the notion that the A756G mutation affects the transfer of the proton stabilizing the leaving group through a set of contacts that locks the phosphate group, greatly increasing the required activation energy.

The catalytic mechanism of the HdV ribozyme was also investigated following a mutational approach. After publication of the initial crystal structure of the product form of the ribozyme, a construct corresponding to a precursor form of the HdV ribozyme bearing a C75 to U mutation and two additional nucleotides at the 5' -end was studied. Ten crystal structures of this construct were solved in the presence of various divalent cations including magnesium, calcium, strontium, barium, manganese, cobalt, copper and iridium in order to identify both the two players of the acid/base reaction [START_REF] Ke | A conformational switch controls hepatitis delta virus ribozyme catalysis[END_REF]. The C75 to U mutation prevents the de-protonation of the N3 position, thus inactivating the ribozyme. The crystal structures show the presence of an ion in the vicinity of the nucleophile. The model, which could be derived from these complementary studies, is that the cation acts as the general base, abstracting the proton from the 2'-hydroxyl nucleophile U-1, and C75 acts as the general acid, stabilizing the 5'-oxyanion of G1. Another crystal structure containing three consecutive deoxyresidues at the 5'-end of the substrate strand was also published [START_REF] Chen | A 1.9 A crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage[END_REF]. The original crystal structures of the product form [START_REF] Ferré-D'amaré | Crystal structure of a hepatitis delta virus ribozyme[END_REF] and of the C75U mutated construct [START_REF] Ke | A conformational switch controls hepatitis delta virus ribozyme catalysis[END_REF] were subject to refinement using the ERRASER program suite in order to rule out structural ambiguities that led to a better interpretation of the catalytic mechanism [START_REF] Kapral | New tools provide a second look at HDV ribozyme structure, dynamics and cleavage[END_REF].

Recently discovered ribozymes

In 2015, a set of new ribozymes was identified by comparative genomics analysis [START_REF] Roth | A widespread self-cleaving ribozyme class is revealed by bioinformatics[END_REF][START_REF] Weinberg | New classes of self-cleaving ribozymes revealed by comparative genomics analysis[END_REF]. These ribozymes were named after the features of their secondary structures.

Twister is "twisted" by a double pseudoknot, like Twister-sister. Pistol and Hatchett look alike these objects. These ribozymes constitute additional families of endonucleolytic ribozymes, which also yield 5'-hydroxyl and 2',3'-cyclic phosphodiester ends. With the benefit of the acquired knowledge on the crystallization of ribozymes, it took only few years to get crystal structures for most of them. This paragraph is dedicated to those ribozymes and to the strategies that were used to crystallize them.

The Twister ribozyme

The Twister ribozyme [START_REF] Roth | A widespread self-cleaving ribozyme class is revealed by bioinformatics[END_REF] is widespread within bacteria, fungi, plants and animals. It seems to be important in genetic control, although such a function has not been demonstrated yet. The ~50 nt minimal Twister ribozyme secondary structure is composed of three helices of about four base pairs separated by two internal loops. The crystal structure of the Oryza sativa Twister ribozyme illustrates how the ribozyme folds [START_REF] Liu | Crystal structure and mechanistic investigation of the twister ribozyme[END_REF]. Basically, the second internal loop adopts a sharp kink allowing stems 2 and 3 to end up side-by-side in a parallel orientation stabilized by the formation of a first pseudoknot (T1) between the apical loop and the 3'-strand of the first internal loop (L1). This conformation is further stabilized by a second pseudoknot (T2) resulting from the interaction of the 3'-nucleotides from the apical loop with the 5'-strand of the second internal loop (L2). The 5'-strand of the first internal loop contains a UA step where the cleavage occurs. T1 is instrumental in positioning the scissile adenosine residue (Figure 9a,b).

In the construct crystallized by the group of David Lilley [START_REF] Liu | Crystal structure and mechanistic investigation of the twister ribozyme[END_REF], catalysis was prevented by the incorporation of a 2'-deoxyuridine (dU) in an RNA entirely chemically synthesized. A crystal packing contact involving the dU perturbs its orientation and is invoked to prevent the observation of the SN2-characteristic O2'-P-O5' in-line orientation. The likeliness of an acido-basic based catalytic mechanism was demonstrated by a combination of molecular modeling studies and pH-dependent catalytic measurements of mutated ribozymes. These studies point to an acido-basic cleavage mechanism, like most nucleolytic ribozymes, with G45 as a general base, since a G45 to A mutation shifts the pKa of the reaction. The position of A29 in the crystal structure could not explain thoroughly the acid pKa observed in catalytic measurement experiments, leaving open the question of its involvement. Two other crystal structures of Twister ribozymes from O. sativa and from an environmental sample without identification of the organism of origin (env) were solved but at lower resolutions and with geometries not permitting to decipher the catalytic mechanism [START_REF] Eiler | Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme[END_REF].

An additional crystal structure from the group of Patel describing the structure of another environmental Twister ribozyme (env22) displays a conformation where a modeled O2' could be properly aligned with the -P-O5' scissile bond [START_REF] Ren | In-line alignment and Mg(2)(+) coordination at the cleavage site of the env22 twister ribozyme[END_REF]. This construct was formed by hybridization of two RNA fragments of 19 and 37 nucleotides obtained by chemical synthesis. The 19-mer contained a dU residue at position 5 to prevent phosphotransfer at residue 6. In this structure, dU5 does not contribute to packing, which rationalizes its better orientation. The structure also confirms the involvement of G48 (equivalent to O. sativa G45) in the acid/base reaction through interaction of its N1group with a non-bridging oxygen atom of the scissile phosphate. The structure also presents a hexa-coordinated magnesium ion, that seems to be involved in catalysis.

Following up, the structure of a mini-Twister ribozyme derived from the env22 construct was solved, in which stem P1 was depleted, since in-solution studies indicated it was dispensable for activity [START_REF] Kosutic | A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class[END_REF]. This construct was inactivated by 2'-Omethylation of U5. The conformation of the scissile dinucleotide is very close to the one observed in the structure containing a dC5 [START_REF] Ren | In-line alignment and Mg(2)(+) coordination at the cleavage site of the env22 twister ribozyme[END_REF], although the atoms involved in the SN2like mechanism are more off-line than in the dU5 substituted ribozyme, a situation apparently due to the bulkiness of the methyl group that influences the pucker of the ribose ring. Nevertheless, in-depth biochemical studies demonstrated the catalytic role of the magnesium ion coordinating the scissile phosphate as well as of A6. Even-though more studies are necessary to precisely identify the role of each actor, the situation is now that Twister seems to adopt a mixed strategy based on metal ion catalysis on the one hand, and on nucleotide-driven acid/base catalysis on the other hand [START_REF] Kosutic | A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class[END_REF].

The twister sister ribozyme

The Twister Sister (TS) ribozyme [START_REF] Weinberg | New classes of self-cleaving ribozymes revealed by comparative genomics analysis[END_REF] was named after secondary structure features that look alike those of the Twister ribozyme. However, its 3D structure [START_REF] Liu | The structure of a nucleolytic ribozyme that employs a catalytic metal ion[END_REF] departs significantly from Twister's, notably by a cleavage site on the 3'-side of the first helix, which forms a distinctive catalytic pocket based on a metal ion with a putative role as a general base activating the nucleophile during catalysis. The 3D structure of the TS ribozyme is organized around two helical stacks weaving tertiary interactions resulting from the 180° rotation of one helical segment with respect to the other, leading to contacts between the apical loop (L4) and the first internal loop (L1). This twist forms a 3-way junction locked by docking of a couple of unpaired nucleotides in specific receptors through stacking interactions and hydrogen bonding (Figure 9c,d). Like in Twister, L4 is responsible for interactions with the two internal loops, L1 and L2. It provides a receptor for A8, which donates its sugar edge to the sugar edges of G23 and gets stacked in between A25 and A26. G27, the ultimate nucleotide of the apical loop is stretched by the backbone rotamers to reach the most distal region to base pair with C14, providing helical continuity between P2 and A15 from the 3-way junction. In L1, C54 and A55, which comprise the scissile bond, adopt a quasi-helical conformation and appear somehow disconnected from the ribozyme core, except by interaction with a magnesium ion having the O2-atom of C54 in its first shell of hydration. The helical conformation of the nucleotides at the scissile bond is not compatible with the S N2-like mechanism and indicates that the structure [START_REF] Liu | The structure of a nucleolytic ribozyme that employs a catalytic metal ion[END_REF] corresponds to a pre-catalytic state.

The RNA strand design strategy was based like in the case of Twister on the depletion of the nucleophilic 2'-hydroxyl group by chemical synthesis of two distinct strands. A ribozyme 40-mer incorporated two 5-bromocytidines, in order to solve the phase problem by anomalous diffraction methods, and a substrate 22-mer incorporated the 2'deoxycytidine.

In another structure of a TS ribozyme solved by the group of Patel [START_REF] Zheng | Structure-based insights into self-cleavage by a four-way junctional twister-sister ribozyme[END_REF], the architecture is based on a 4-way junction and not on a 3-way junction like in the above example. The structure was solved by heavy atom anomalous diffraction methods using iridium hexammine. The 4-way junction as well as an extended L1 internal loop forms a significantly different organization of the overall structure and around the catalytic site.

Firstly, the additional stem P3 extends the helical stack formed by P1 and P2 generating a 4-way junction where the angle between the two helical stacks (P1-P2-P3 and P4-P5) is close to 90°. The expanded L1 provides opportunity for a set of non-canonical base pairs, which also chelate several magnesium ions, in close vicinity of the catalytic site. In spite of a similar strategy was applied to deplete the nucleophile by chemical synthesis of a ribozyme and of a substrate strand incorporating the required modification, the two nucleotides around the scissile bond do not adopt a helical conformation. While L4 serves as a receptor for residue A7 (equivalent to A8 from 5t5a), G35 (equivalent to G27 from 5t5a) interacts with a magnesium ion, which itself is connected to the backbone of the 5'-strand of L1. The intricate set of interactions between L1 and L4 residues seems to promote specific contacts within the purine-rich L1 that lead to the extrusion of the 2'-deoxycytidine residue (C62) in a conformation that could favor the SN2-like reaction, since A63 is clamped within the helix by means of hydrogen bonds and stacking interactions locked by magnesium ions. Surprisingly, no ion is observed bridging the A residues from L4 with the groove of L1 as observed in the structure from the group of Lilley [START_REF] Liu | The structure of a nucleolytic ribozyme that employs a catalytic metal ion[END_REF].

In spite of the application of the same strategy to inactivate the nucleophile by depletion of the 2' -hydroxyl group, these two structures present very different features. The interpretation of the catalytic mechanism needs to be reinforced in one case (5t5a) with specific biochemical studies, whereas in the other case (5y85) the interpretation is more straightforward directly from the structure. The sequences of the ribozymes themselves seem to be responsible for these observations and point to the difficulty to yield exploitable structural data to shed light on catalytic mechanisms.

The Pistol ribozyme

Crystal structures of the Pistol ribozyme were solved quickly after its discovery and the first functional studies [START_REF] Ren | Pistol ribozyme adopts a pseudoknot fold facilitating sitespecific in-line cleavage[END_REF][START_REF] Nguyen | Crystal structure of Pistol, a class of self-cleaving ribozyme[END_REF]. The env25 ribozyme [START_REF] Ren | Pistol ribozyme adopts a pseudoknot fold facilitating sitespecific in-line cleavage[END_REF] was made up of two chemically synthesized annealed strands, an 11-mer substrate strand, and a 47-mer ribozyme core.

The env27 ribozyme was made up of a 15 nt substrate, while the core was obtained by in vitro transcription [START_REF] Nguyen | Crystal structure of Pistol, a class of self-cleaving ribozyme[END_REF]. In both cases, cleavage was prevented by incorporation of a deoxy residue in place of G53 (env25) or G10 (env27). The secondary structure is composed of a short hairpin followed by a longer one. The latter is interrupted by a very asymmetric internal loop, the longest strand of which forms a half-helical turn pseudoknot with the loop of the former hairpin. In the 3D structure, the pseudoknot is clamped between stems P1 and P3, making an extended helical core. The strand connecting P1 to P2 elegantly follows the shallow groove of P1 and sets P2 off the ribozyme core. Since the final structures showed very similar features, we describe details from the env25 structure. This arrangement, together with the constraints due to the formation of the P2 helix, produces the sharp kink necessary to splay apart the nucleotides around the scissile phosphate group of C54 (Figure 6.2.10). Initial biochemical analysis [START_REF] Harris | Biochemical analysis of pistol self-cleaving ribozymes[END_REF], NMR and fluorescence spectroscopy data obtained on the crystallized forms of the ribozymes point to the N3 group of A32 to be responsible for stabilization of the 5'-oxyanion leaving group, while G40 would be able to activate the nucleophile. The dG modification does not prevent to observe a magnesium ion close to the scissile phosphate. However, this ion seems to play a structural role rather than a catalytic one, since it is not directly coordinated to any RNA atom.

Conclusions and perspectives

All the examples reported in this review show how structural models offer a powerful heuristic framework to design and interpret the results of biochemical and mutational studies toward a better understanding of the catalytic mechanisms of ribozymes. This means that even-though significant knowledge is required to inactivate the ribozyme to crystallize, it is from the structures obtained that the conclusive experiments may be designed. Efficient and diverse strategies for controlling ribozyme catalysis in the context of crystallization studies is thus a valuable addition to the biochemist's toolbox.

In these strategies, the solid phase chemical synthesis of oligonucleotides holds a central role.

Technical progresses in chemical and enzymatic synthesis, in heavy atom derivatization of RNA, in softwares both for RNA folding predictions and crystal structure refinement as well as data collection and treatment, and in detector sensitivity allow for solving RNA structures always faster. The main bottleneck remains crystallization, since the stability of an RNA molecule does not only depend on its architecture, but also on its folding process, as well as on the crystal packing. In the future, x-ray free electron laser (XFEL) facilities may help to release the pressure on obtaining large crystals, as in a recent study, in which distinct forms of a riboswitch could be observed simultaneously, [START_REF] Stagno | Structures of riboswitch RNA reaction states by mixand-inject XFEL serial crystallography[END_REF] and thus become a more routine technic.

To summarize, the modifications, which require lesser knowledge on catalysis are the deoxy substitution of the nucleophile or its methylation. Another strategy consists in using a split substrate and results in the loss of the scissile phosphate. All these modifications perturb the catalytic site organization, and structural interpretations should be made with the support of biochemical data obtained on the unmodified ribozyme form. The construction of a circularly permutated ribozyme is always possible and mimics the split substrate strategy. In this case, in vitro transcription can be used to produce a single strand RNA of any length opening up the possibility to purify the RNA of interest under native conditions directly from the transcription mix . Finally, studying the transition state structural mimic using a vanadate ion constitutes a fourth strategy that can be added on top of the split substrate or circular permutation. When nucleotide candidates involved in nucleophile activation or leaving group stabilization are identified, it is also possible to design mutated constructs with very poor catalytic properties.

1 6. 2

 12 Strategies for crystallization of natural ribozymes B. Masquida*, D. Sibrikova, Maria Costa 6.2.1 Introduction

  2.5a), which undergoes a nucleophilic attack at the 5'-splice site. This first splicing step results in the formation of a specific branched intron intermediate, the 'lariat' intermediate, in which the 'branchpoint' adenosine (bpA) becomes covalently linked to the first intron nucleotide, a conserved guanosine, through a 2',5'-phosphodiester bond. It follows the second transesterification reaction, during which the 3'-OH of the cleaved 5'-exon attacks the 3'-splice site, resulting in the ligation of the flanking exons and the release of the intron 'lariat'. Because transesterification reactions are chemically reversible, excised group II lariats are then able to invade specific RNA or DNA targets by catalyzing the splicing reactions in the reverse order and orientation: this 'reverse splicing' pathway plays an essential role in the biological activities of group II introns , since reverse splicing into DNA triggers the genomic mobility of the intron.

6. 2 . 9

 29 Acknowledgement BM is supported by CNRS and by the LabEx MitoCross (LABEX ANR-11-LABX-0057_MITOCROSS). DS is supported by an IdEx doctoral grant obtained from the University of Strasbourg (BGPC 17/N° ARC 26349). MC acknowledges funding from BIG LidEx program.

Figure 6 . 2 . 1 :

 621 Figure 6.2.1: The folded core of the HdV (a) and GlmS (b) ribozymes corresponds to the

Figure 6 . 2 . 2 :

 622 Figure 6.2.2: The GlmS riboswitch is also a ribozyme. (a) The GlmS riboswitch does not change drastically its conformation upon binding of the GlcN6P. Rather, it undergoes

Figure 6 . 2 . 3 :

 623 Figure 6.2.3: This figure displays the structures of the minimal (pdb: 1mme) (a) and the extended (pdb: 3zd5) (c) versions of the hammerhead ribozyme. Panels (b) and (d)

Figure 6 . 2 . 4 :

 624 Figure 6.2.4: (a) Splicing pathway mediated by the group I introns in general (see text for details). (b) Secondary structure depicting the tertiary interactions observed in the

Figure 6 . 2 . 5 :

 625 Figure 6.2.5: Structure of a group II intron lariat primed for reverse splicing. (a) Secondary structure outline, colored by domains, of the crystallized group II intron

Figure 6 . 2 . 6 :

 626 Figure 6.2.6: The hairpin ribozyme can be active as a short or an extended version, provided that the internal loops of domains A and B establish specific interactions. In the

Figure 6 . 2 . 7 :

 627 Figure 6.2.7: Organization of the LC ribozyme constructs. (a) Representation of the

Figure 6 . 2 . 8 :

 628 Figure 6.2.8: The secondary structure of the crystallized construct of the VS ribozyme

Figure 6 . 2 . 9 :

 629 Figure 6.2.9: The Twister (a, b) and Twister Sister (TS) (c, d) ribozymes. Inspite of similarities at the secondary structure level, these two ribozymes adopt distinct

Figure 6 . 2 . 10 :

 6210 Figure 6.2.10: Structural aspects of the Pistol ribozyme (pdb 5k7e). (a) The secondary