Mirijam-Elisabeth Zeller

Agnes Csanadi

Andras Miczak

Thierry Rose

Thierry Bizebard

Vladimir R Kaberdin
email: vladimir.kaberdin@univie.ac.at

Quaternary structure and biochemical properties of mycobacterial RNase E/G

Keywords: M. tuberculosis, ribonuclease, 5´-end-dependence, RNA processing

The RNase E/G family of endoribonucleases plays the central role in numerous posttranscriptional mechanisms in E. coli and, presumably, in other bacteria including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of M.

tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. Here, we show that affinity-purified MycRne has a propensity to form di-and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5´-phosphorylation status of RNA. Our data also indicate that the cleavage specificities of the M. tuberculosis RNase E/G homologue and its E. coli counterpart are only moderately overlapping and reveal a number of sequence determinants within MycRne cleavage sites that differentially affect the efficiency of cleavage. Finally, we demonstrate that, similar to E. coli RNase E, MycRne is able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA, thus suggesting a common function for RNase E/G homologues in rRNA processing.

INTRODUCTION

The RNase E/G family of endoribonucleases is known for its essential role in RNA processing and decay in bacteria [START_REF] Cohen | RNase E: Still a wonderfully mysterious enzyme[END_REF][START_REF] Coburn | Degradation of mRNA in Escherichia coli: An old problem with some new twists[END_REF][START_REF] Régnier | Degradation of mRNA in bacteria: emergence of ubiquitous features[END_REF][START_REF] Kushner | mRNA decay in Escherichia coli comes of age[END_REF]. Our current knowledge about these enzymes mainly originates from studies of E. coli RNase G and RNase E, site-specific ribonucleases that preferentially cleave in A/U-rich single-stranded regions of structured RNAs.

E. coli

RNase E is encoded by the rne gene and is indispensable for cell viability [START_REF] Deana | The function of RNase G in Escherichia coli is constrained by its amino and carboxyl termini[END_REF][START_REF] Ow | RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue, RNase E[END_REF]. Structural data obtained for an N-terminal polypeptide representing the evolutionarily conserved catalytic domain of this protein (Fig. 1) have revealed the presence of discrete folds with putative functions in RNA recognition and cleavage [START_REF] Callaghan | Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover[END_REF]. Moreover, these data and others [START_REF] Callaghan | Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain[END_REF][START_REF] Callaghan | Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease[END_REF] indicate that the catalytic domain exists as a homotetramer, which has two nonequivalent subunit interfaces organizing the active site of the enzyme. In contrast to the N-terminal catalytic domain, the C-terminal part of E. coli RNase E shows very little or no similarity to the equivalent regions of other RNase E homologues [START_REF] Kaberdin | The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the Cterminal half, which is sufficient for degradosome assembly[END_REF]. This part of the E. coli enzyme contains an extra RNA-binding domain(s) [START_REF] Leroy | Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA[END_REF]12] and multiple sites for interactions with the 3´ to 5´ phosphorolytic exonuclease polynucleotide phosphorylase (PNPase), the RNA helicase RhlB and the glycolytic enzyme enolase [START_REF] Kaberdin | The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the Cterminal half, which is sufficient for degradosome assembly[END_REF]13] to form the E. coli RNA degradosome [14].

E. coli

RNase G [15,16] is encoded by the rng gene and was initially termed CafA due to its effects on the formation of cytoplasmic axial filaments observed upon RNase G overproduction in vivo [17,18]. Further studies revealed that this protein has an endoribonucleolytic activity [19] and is involved in maturation of 16S rRNA in vivo [15,16].

Although RNase G functionally overlaps with RNase E and shares 35% identity and 50% similarity with the N-terminal catalytic part of RNase E [20], this endoribonuclease cannot fully compensate RNase E deficiency in rne ts mutant strains [START_REF] Deana | The function of RNase G in Escherichia coli is constrained by its amino and carboxyl termini[END_REF][START_REF] Ow | RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue, RNase E[END_REF].

Due to their overlapping functions and apparently common origin RNase E-and

RNase G-like proteins are believed to belong to the same family of RNase E/G endoribonucleases that are predicted to exist in many bacteria including pathogenic species [21][22][23]. Although their biochemical properties and biological functions have not yet been investigated in detail in human pathogens, such studies can offer important insights concerning the role of these endoribonucleases during infection and disease development.

This consideration prompted us to purify and characterize the RNase E/G homologue (MycRne) from the intracellular pathogen M. tuberculosis.

We have shown that, similar to E. coli RNase G [START_REF] Briant | The quaternary structure of RNase G from Escherichia coli[END_REF] and the catalytic domain of E.

coli RNase E [START_REF] Callaghan | Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain[END_REF][START_REF] Callaghan | Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease[END_REF], MycRne is a 5´-end-dependent endoribonuclease, which can exist in di-E. coli cells containing pMtRne plasmid were grown at 37°C in LB supplemented with ampicillin (100 µg/ml), chloramphenicol (34 µg/ml) and glucose (0.75%). At an OD 600 ~0.5, the cell culture was pre-cooled on ice for 45 min, and protein expression was induced by addition of IPTG to 1 mM and incubated for 16 h at 15°C further. Cells were harvested by centrifugation (15 min, 7000 g), frozen at -20°C, thawed and suspended in ice-cold binding buffer (0.5 M NaCl, 20 mM sodium phosphate, pH 7.4) supplemented with pepstatin (1 µg/ml) and protease inhibitor cocktail (1 tablet per 3 g of wet cells) (Roche). The cells were disrupted by sonication in a Cell Disruptor W375 (Heat Systems Ultrasonics Inc.), the cell lysate was further cleared by centrifugation (15 min, 16000 g, 4°C) and the supernatant was applied to a HisTrap HP (Amersham Biosciences) column to purify 6His-tagged MycRne according to the vendor's instructions. Column fractions containing MycRne were combined, supplemented with EDTA and DTT to final concentrations of 0.1 mM and 1 mM respectively, dialyzed extensively against EB buffer (50 mM NaCl, 20 mM HEPES, pH 7.5, 1 mM DTT, 0.1 mM EDTA) and further stored in small aliquots at -80°C.

Gel filtration

Affinity-purified MycRne was dialyzed against low-salt buffer (75 mM NaCl, 10 mM HEPES, pH 7.5, 1 mM DTT, 0.1 mM EDTA) and concentrated to 8.25 mg/ml employing a Centricon YM-30 device (Millipore). The concentration of MycRne was calculated based on its absorbance at 280 nm and using extinction coefficients estimated from its amino acid composition by ProtParam (ExPaSy server, www.expasy.org). Prior to loading an aliquot (~150 µl) of the concentrated protein sample on a 25-ml Superose 12 HR10/30 column (Amersham Biosciences), the column was pre-equilibrated with low-salt buffer and calibrated using molecular weight markers (aldolase (158 kDa), BSA (67 kDa) and chymotrypsinogen A (25 kDa)). The column was run using the same buffer, and 0.5 ml-fractions were subsequently collected and analyzed by spectrophotometry and SDS-polyacrylamide gel electrophoresis.

Analytical ultracentrifugation

An aliquot of affinity-purified MycRne was dialyzed against 20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and further used in sedimentation equilibrium experiments, which were performed at 20°C on a Beckman Optima XL-A ultracentrifuge with an AN60-Ti rotor. The same buffer was used to dilute samples and as a blank. Long runs were performed at 6 speeds (5, 6, 7, 9. Copyright 2007 Biochemical Society whereas short runs were processed at 10 speeds (5, 6, 7, 9.5, 12, 14, 16, 18, 20 and 45 krpm) using 40 µl samples at 3 concentrations (84 µM, 42 µM and 16 µM) analysed at 297, 292 and 286 nm, respectively. Absorbance analysis (220 to 320 nm) revealed that the equilibrium at 5 krpm was reached after 2 and 18 hours for 40 µl and 150 µl samples, respectively. Absorbance profiles were recorded 5 times with a step of 0.001 cm for every speed and fitted simultaneously on single species or self-association models. The molar extinction coefficient at 280 nm was computed from the protein sequence as ε 280 = 33200 M -1 cm -1 [START_REF] Pace | How to measure and predict the molar absorption coefficient of a protein[END_REF] and extrapolated from protein spectra at other wavelengths (ε 233 = 91721 M -1 cm -1 , ε 292 = 12025 M -1 cm -1 , ε 297 = 2886 M -1 cm -1 ). Variances between experimental points and best fits were used to select the models. The MycRne specific partial volume v-bar = 0.713 mL/g was computed from the protein sequence.

In vitro transcription and labelling of 9S RNA

A DNA fragment including the putative Mycobacterium bovis BCG 9S RNA sequence was PCR amplified from genomic DNA using primers 9Sfor (5´-TGCTAACCGGCCGAAAACTTA-3´) and 9Srev (5´-AACATACAAAAACACCACCGT-3´), and cloned into the pGEM ® -T Easy vector (Promega) at the T cloning site. The resulting plasmid (pMt9S) was further verified by sequencing, linearized by PstI (MBI), blunted with Klenow enzyme (MBI), purified in a 1% agarose gel and used to transcribe 9S RNA with a MEGAscript ® T7 kit (Ambion). 9S RNA was further purified from a 6% polyacrylamide /urea gel, dephosphorylated with bacterial alkaline phosphatase (MBI Fermentas) and 5´labelled using T4 polynucleotide phosphorylase (MBI Fermentas) and an excess of [γ-32 P] ATP (Amersham). 5´-end labelled 9S RNA was further purified from a 6% (w/v) polyacrylamide sequencing gel as described [START_REF] Mcdowall | Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops[END_REF] and subsequently used for cleavage assays.

Primer extension

Total RNA from Mycobacterium bovis BCG was extracted using the hot phenol method [START_REF] Lin-Chao | Effect of the bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli[END_REF]. Copyright 2007 Biochemical Society analyzed on an 8% sequencing gel along with a concurrently run sequencing ladder. The latter was prepared using PstI-digested pMt9S, the same 5'-[ 32 P]-labelled primer and a Reader TM DNA sequencing kit (MBI Fermentas).

Synthetic oligonucleotide substrates

Ribooligonucleotides 5´-ACAGUAUUUG (BR10), 5´-ACAGAAUUUG (9SA) 5´-GAAGGAUUUAA (OmpC), 5´-AAAAAAAAAAAAAAAAAAAAAAAAAAA (A27), 5´-UUUUUUUUUUUUUUUUUUUUUUUUUUU (U27), 5´-UUUUUUUUUUUUUGUUUU-UUUUUUUUU (U27G), 5´-UUUUUUUUUUUUUAUUUUUUUUUUUUU (U27A), 5´-UUUUUUUUUUUUUCUUUUUUUUUUUUU (U27C) as well as oligonucleotides 5´-pGGGACAGUAUUUG (pBR13-Fluor) and 5´-HOGGGACAGUAUUUG (HO-BR13-Fluor) with a fluorescein tag at their 3´ ends were purchased from VBC Genomics (Vienna), whereas UUUUUUUUUUUUUXUUUUUUUUUUUUU (U27Ab) containing abasic residue (X) was purchased from Dharmacon (USA).

RNase E/G cleavage of oligonucleotide substrates and 9S RNA

RNase E/G cleavage reactions were performed as described previously [START_REF] Mcdowall | Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops[END_REF] using Rne498 (or MycRne) and 5´-labelled substrates. 1 nt ladders were prepared either by partial alkaline hydrolysis of each 5´-end labelled substrate in 1mM EDTA, 50 mM sodium carbonate (pH

RESULTS

Cloning and purification of M. tuberculosis RNase E/G

To learn more about organization and biochemical properties of RNase E/G from Grampositive bacteria, the M. tuberculosis RNase E/G homologue was overproduced in Copyright 2007 Biochemical Society into an expression vector and verified by sequencing. The recombinant MycRne carrying a 6His-tag was overexpressed in E. coli strain BL21-CodonPlus(DE3)-RIL and purified under native conditions. As seen in Fig. 1B, the 71 kDa MycRne protein exhibited an aberrant mobility, migrating as a 110 kDa polypeptide. Slower running of RNase E/G homologues in SDS polyacrylamide gels has been reported previously and appears to be caused by their unusual amino acid composition [START_REF] Casaregola | Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein[END_REF]. Mass spectrometry analysis did not reveal any contamination of the MycRne preparation by E. coli RNase E/G polypeptides or other host ribonucleases (data not shown).

Quaternary structure of M. tuberculosis RNase E/G

The putative catalytic domain of MycRne contains the highly conserved Zn-link motif known to be implicated in di-and tetramerization of E. coli RNase E/G [START_REF] Callaghan | Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain[END_REF][START_REF] Callaghan | Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease[END_REF]. To assess whether the ability of RNase E/G homologues to form di-and tetrameric forms is evolutionarily conserved, we analyzed the oligomerization state of affininy-purified MycRne. According to the calibration profile obtained with aldolase, BSA and chymotrypsinogen A, MycRne was eluted as a complex with a hydrodynamic radius corresponding to a spherical protein with an apparent molecular mass of approximately 325 kDa (Fig. 2A), suggesting that, similar to E. coli homologues, MycRne may exist in a tetrameric form.

In order to confirm the oligomerization state of MycRne by an independent method, we performed analytical ultracentrifugation (Fig. 2B). [START_REF] Mcdowall | Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops[END_REF][START_REF] Afonyushkin | Temperaturedependent stability and translation of Escherichia coli ompA mRNA[END_REF][START_REF] Kaberdin | Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E[END_REF]. As seen in Fig. 3, MycRne could cut all three oligonucleotides yielding cleavage patterns that were largely indistinguishable from the analogous patterns generated by its E. coli counterpart. A minor difference was only observed in the cleavage pattern of OmpC. Although Rne498 cleaved this oligonucleotide at positions 5 and 6 with equal efficiencies, MycRne showed some preference for cleavage at position 5.

We also found that, similar to its RNase E/G homologues from A. aeolicus and E. coli, MycRne requires magnesium ions for its activity (Fig. 3E).

5´-end dependence of M. tuberculosis RNase E/G cleavages

RNase E/G homologues from Gram-negative bacteria are known to preferentially cleave 5´monophosphorylated substrates when compared to non-phosphorylated [19, [START_REF] Kaberdin | Characterization of Aquifex aeolicus RNase E/G[END_REF][START_REF] Redko | Determination of the catalytic parameters of the N-terminal half of E. coli ribonuclease E and the identification of critical functional groups in RNA substrates[END_REF] or 5´triphosphorylated ones [START_REF] Mackie | Ribonuclease E is a 5'-end-dependent endonuclease[END_REF]. Employing 5´-monophosphorylated and non-phosphorylated BR13 (5´-GGGACAGUAUUUG-3´) tagged with a fluorescein group at their 3´ ends, we demonstrated that, similar to its counterparts, MycRng cleaves 5´-monophosphorylated substrates faster than the non-phosphorylated ones (Fig. 4A andB). Thus, our data suggest that the so-called 5´-end dependence is apparently a common feature of RNase E/G homologues from both Gram-negative and Gram-positive bacteria.

Probing the substrate specificity of M. tuberculosis RNase E/G

In order to identify specific sequence determinants of MycRne cleavage sites we used essentially the same oligonucleotide-based approach that was previously employed to characterize its E. coli counterpart [35]. By comparing the cleavage patterns of poly(A) and poly(U) oligonucleotides and their mutant variants carrying single-base substitutions of adenosine (or uridine) by G, C, U or A, respectively, this method allows determining the effect of each nucleotide at multiple positions to the point of cleavage [35].

Unlike E. coli RNase E [START_REF] Huang | Poly(A)-and poly(U)-specific RNA 3' tail shortening by E. coli ribonuclease E[END_REF][START_REF] Walsh | Cleavage of poly(A) tails on the 3´-end of RNA by ribonuclease E of Escherichia coli[END_REF], MycRne cleaved A27 inefficiently. As seen in Fig. 5A, despite 5 fold excess of MycRne (when compared to the cleavage conditions of U27), A27 cleavage products can be readily detected only on overexposed X-ray films (Fig. 5A). In the following assays, we therefore decided to use only U27 (control) and its derivatives, U27A, U27G, U27C and U27ab oligonucleotides, that had a single base substitution of U at position 14 by either A, G, C or abasic residue, respectively. 9

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

Copyright 2007 Biochemical Society Similar to cleavage of U27 (Fig. 5B), incubation of U27G with MycRne and Rne498 resulted in nearly identical patterns (Fig. 6A). The major cleavages of U27G at positions U8, U9, U10 and U15 were observed for both enzymes, whereas Rne498 (but not MycRne) could additionally cleave this substrate at position U18. In the case of E. coli RNase E, it has been previously reported that a G nucleotide, which is 5´ and in close vicinity to the scissile bond, is an important determinant of cleavage [START_REF] Kaberdin | Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E[END_REF][START_REF] Redko | Determination of the catalytic parameters of the N-terminal half of E. coli ribonuclease E and the identification of critical functional groups in RNA substrates[END_REF]35]. Likewise, we found that M. tuberculosis RNase E/G cleavage is more efficient if there is a G nucleotide located 2 nucleotides upstream of the scissile bond (Fig. 6A). These data are fully consistent with the cleavage patterns of BR10, 9SA and OmpC oligonucleotides showing that MycRne cleavage frequently occurs 2 nucleotides downstream of a G nucleotide (Fig. 3).

In contrast to nearly identical cleavage patterns that were generated by MycRne and Rne498 using U27G (Fig. 6A), the cleavage patterns of U27A, U27C and U27ab showed relatively low resemblance (Fig. 6B, C and D, respectively). Although Rne498 can cut at multiple positions within U27A (Fig. 6B), MycRne cleavage of the same substrate was not observed at many of these locations, especially downstream and upstream of U16. Similarly, the presence of a cytosine at position 14 in U27C (Fig. 6C) strongly inhibits MycRne (and to a lesser degree Rne498) cleavage at nearly all locations suggesting the overall inhibiting effect of this nucleotide. Finally, we found that substitution of the U at position 14 by an abasic residue in U27ab (Fig. 6D) decreased the efficiency of MycRne cleavage at multiple locations. This finding strongly suggests that the presence of uridines at many locations close to the scissile bond promotes MycRne cleavage.

Based on the above experimental data, we determined and summarized the effects of each nucleotide within MycRne cleavage sites (Fig. 6E). Interestingly, according to these data, the C at position +2 in 9SA should confer higher resistance of this oligonucleotide (in comparison to BR10 lacking a cytosine at the equivalent position) to MycRne. In contrast, we did not observe any significant differences in the efficiency of cleavage (Fig. 3, unpublished data). This may imply that the expected inhibiting effect of the cytosine was efficiently counteracted by other nucleotides, for example, by the A and G nucleotides at positions - Copyright 2007 Biochemical Society 5´ to the scissile bond (the "end-proximity effect"), thereby leading to accumulation of short oligomers that are further resistant to the nucleolytic activity of this enzyme.

9S RNA processing by M. tuberculosis RNase E/G

While playing an essential role in maturation of stable RNA in E. coli, RNase E is known to process the 9S RNA precursor of the 5S rRNA [START_REF] Ghora | Structural analysis and in vitro processing to p5 rRNA of a 9S RNA molecule isolated from an rne mutant of E. coli[END_REF]. To find out whether MycRne might act likewise in the processing of rRNA in M. tuberculosis, we examined the cleavage pattern obtained after incubation of in vitro transcribed M. tuberculosis 9S RNA with Rne498 and MycRne. Although both enzymes could cleave this RNA, the cleavages occurred at different locations (Fig. 7A, on the left). This finding is consistent with our data (Fig. 6) suggesting some differences in the substrate specificities of E. coli and M. tuberculosis RNase E/G homologues. Further analysis (Fig. 7B, on the right) revealed that MycRng cleaved 9S RNA at the position, which nearly coincides with the position of the 5´ end of mature 5S rRNA in vivo, which was mapped by primer extension of total RNA (Fig. 7B andC).

DISCUSSION

Mycobacteria are Gram-positive bacteria known to enter a state of metabolic dormancy when they encounter anoxic or nutritionally deprived environments [START_REF] Cardona | On the nature of Mycobacterium tuberculosis-latent bacilli[END_REF]. This mechanism allows pathogenic species such as M. tuberculosis to survive within their host-cells and involves numerous changes in gene expression to convey the required physiological adjustments.

Adaptive processes in the RNA metabolism of latent mycobacteria differentially affect the steady-state levels of numerous transcripts [START_REF] Schnappinger | Transcriptional adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the phagosomal environment[END_REF], ribosome biosynthesis [START_REF] Beste | Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism[END_REF] as well as RNase E/G level [START_REF] Archuleta | Mycobacterium avium enters a state of metabolic dormancy in response to starvation[END_REF], suggesting an important regulatory role of this enzyme during the disease development. To learn more about the function and biochemical properties of the RNase E/G homologue from M. tuberculosis (MycRne), we cloned, purified and characterized a polypeptide including the centrally located catalytic domain as well as the flanking C-terminal part of this protein (Fig. 1).

We found that, similar to its homologues, E. coli RNase E (residues 1-530; [START_REF] Callaghan | Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover[END_REF][START_REF] Callaghan | Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain[END_REF][START_REF] Callaghan | Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease[END_REF]) and

RNase G [START_REF] Briant | The quaternary structure of RNase G from Escherichia coli[END_REF] polypeptides, purified MycRne can exist in solution in di-or tetrameric forms.

Previous work has shown that a pair of cysteine residues in the conserved CPxCxGxG motif, which coordinates a zinc ion (so-called Zn-link motif), is critical for assembly of the E. coli RNase E homotetramers [START_REF] Callaghan | Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover[END_REF]. Given that the Zn-link motif exists in MycRne and other members of the RNase E/G family, our results suggest that the protein-protein interfaces, 11 Copyright 2007 Biochemical Society which are involved in intermolecular association of MycRne (this study) and its E. coli counterparts [START_REF] Callaghan | Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover[END_REF][START_REF] Callaghan | Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain[END_REF][START_REF] Callaghan | Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease[END_REF][START_REF] Briant | The quaternary structure of RNase G from Escherichia coli[END_REF] apparently resemble each other and that the association as dimer-ofdimers may be a common feature of RNase E/G homologues. Interestingly, although the tetrameric form of the catalytic domain of E. coli RNase E (amino acid residues 1-530) is more active in vitro than the monomeric one [START_REF] Callaghan | Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease[END_REF], a truncated E. coli RNase E polypeptide (amino acid residues 1-395), which retains an endoribonucleolytic activity (but has no potential to self-associate), can functionally substitute the full-length E. coli RNase E in vivo [START_REF] Caruthers | Retention of core catalytic functions by a conserved minimal RNase E peptide that lacks the domain required for tetramer formation[END_REF]. Although this finding suggests that bacterial cells can survive with RNase E functioning as a monomer, a tetrameric quaternary structure of this enzyme may be still important to confer efficient cleavage of RNA in fast-growing cells.

By employing oligonucleotide substrates, we have also shown that MycRne has an endoribonucleolytic activity, which is dependent on the 5´ phosphorylation status of its substrates, thereby suggesting that MycRne is a 5´-end-dependent endoribonuclease. This property of MycRne (this study) and other RNase E/G homologues [19,[START_REF] Kaberdin | Characterization of Aquifex aeolicus RNase E/G[END_REF][START_REF] Mackie | Ribonuclease E is a 5'-end-dependent endonuclease[END_REF][START_REF] Jiang | Regions of RNase E important for 5'end-dependent RNA cleavage and autoregulated synthesis[END_REF][START_REF] Lin-Chao | The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo[END_REF] implies that these enzymes more efficiently cleave already damaged or partly degraded forms of cellular RNAs (i.e. RNA species that usually carry 5´-monophosphate groups) rather than primary transcripts, which are naturally triphosphorylated.

The enzyme can cleave short oligonucleotides with the specificity, which is similar to that observed previously for E. coli RNase E (Fig. 3). However, despite their seemingly indistinguishable sequence specificities (Fig. 3), further analysis using homooligomeric substrate U27 and its monosubstituted derivatives U27A, U27C, U27G and U27ab (Fig. 6) revealed a number of important differences in the optimal location of G, C, U and A Copyright 2007 Biochemical Society

Our recent work demonstrate that the knowledge of specific sequence determinants within RNase E/G cleavage sites can be instructive to suggest mutations that increase/decrease the efficiency of cleavage within the site of interest [35] and to infer differences in the substrate specificities of various RNase E/G homologues (this study).

However, this information may not be sufficient per se to predict potential RNase E cleavage sites within a regular transcript. Additional factors such as RNA structure or interactions with RNA-binding proteins should be also taken into consideration.

Given that RNase E and RNase G are involved in maturation of ribosomal and transfer

RNAs in E. coli [START_REF] Misra | RNase E, an RNA processing enzyme from Escherichia coli[END_REF][START_REF] Li | RNase E plays an essential role in the maturation of Escherichia coli tRNA precursors[END_REF], we anticipated that this function of RNase E/G-like proteins might be also conserved in mycobacteria. Indeed, we found that MycRne was able to cleave mycobacterial 9S RNA (Fig. 7A), a putative precursor of its cognate 5S rRNA, and the cleavage occurred in close vicinity to the mature 5´-end of 5S rRNA (Fig. 7B andC), thereby suggesting a role for this enzyme in rRNA processing. Although the internucleotide bonds at the 5´ end of 5S rRNA, that are generated by MycRne in vitro (Fig. 7A) and during rRNA processing in vivo (Fig. 7B) were not exactly the same, this minor (1-2 nucleotide) difference could be well explained by the action of ancilliary factors or associated ribosomal proteins that can affect MycRne cleavage in vivo and, therefore, slightly influence the selection of the scissile bond(s). Further efforts will be necessary to get a better understanding of diverse roles of MycRne in RNA processing and to reveal and characterize the MycRne-mediated, posttranscriptional mechanisms that are involved in adaptive stress responses and the associated alterations in RNA metabolism in mycobacteria.

McDowall, K.J., and Cohen, S.N. (1996)

9. 2 )

 2 at 85 o C for 20 min or by partial digestion of 5´-end labelled oligonucleotides with S1 nuclease in buffer provided by the vendor (MBI). RNase T 1 digests of 9S RNA were prepared by incubation of 5´-end labelled 9S RNA in 10 mM Tris/HCl, pH 8.0; 100 mM sodium chloride, and 5 mM magnesium chloride with RNase T 1 (MBI) at 37 o C.

7 Biochemical

 7 Escherichia coli and purified by affinity chromatography (see Materials and Methods). Since the full-length M. tuberculosis RNase E/G is difficult to purify in large amounts due to its tendency to form insoluble aggregates upon overexpression (unpublished observation), we cloned its truncated form of 621 amino acids, referred to as MycRne. The amino acid sequence of MycRne includes the entire catalytic domain and the adjacent C-terminal region of this protein (Fig.1A). The corresponding genomic fragment was amplified by PCR, ligated Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

Fig. 1 .

 1 Fig. 1. Purification of MycRne. (A) Primary structures of E. coli and M. tuberculosis RNase E/G homologues. The evolutionarily conserved minimal catalytic domain [43] is shown by black boxes in the fulllength of E. coli RNase E, RNase G and M. tuberculosis RNase E/G (MycRne (FL)) polypeptides as well as in the N-terminally truncated form of M. tuberculosis RNase E/G (MycRne) used in this study. The `protein scaffold` region (back-hatched box) of E. coli RNase E (residues 688-1061; [13]) is the location of the binding sites for the major components of the degradosome (enolase, RhlB RNA helicase and polynucleotide phosphorylase (PNPase)). (B) Purification of MycRne. Affinity-purified mycobacterial MycRne as well as protein extracts prepared from BL21 (DE3) cells harbouring the MycRne-coding plasmid before (-) and after (+) addition of IPTG, respectively, were analyzed in 10% SDS gels followed by Coomassie blue staining. Indicated are the positions of M. tuberculosis RNase E/G (MycRne) and the size of several proteins in the molecular weight protein marker (lane M).

Fig. 2 .

 2 Fig. 2. Quaternary structure of RNase E/G.(A) Gel filtration of MycRne. Affinity-purified MycRne (bold curve) was analyzed by sizeexclusion chromatography using a 25 ml-Superose ® 12 HR 10/30 column (Amersham Biosciences) connected to an FPLC system. The column was calibrated with aldolase (158 kDa), BSA (67 kDa) and chymotrypsinogen A (25 kDa) (light curve). Elution profiles were recorded online; the absorbance at 280 nm is indicated on the y-axis, whereas elution volumes are indicated on the x-axis.(B) Sedimentation equilibrium analysis. A representative radial concentration distribution ofRNase E/G at 16 µM is shown after reaching equilibrium (18h) at 12000 rpm at 20ºC. The lower panel plots the absorbance (OD) at 292 nm against the radial position (cm). The absorbance offset was set to 0.022 OD. The best fit of the data set (6 speeds, 1,502 points) was consistent at this sample concentration with a monomer-dimer equilibrium (solid lines) with a K d = 1.8 µM. Residual values between experimental data and a self associating model of ideal species (molecular weight = 71,100 Da) are reported in the upper panel.

Fig. 3 .

 3 Fig. 3. Cleavage patterns of oligonucleotides BR10, 9SA and OmpC. Each 5´ end labelled oligonucleotide (panel A, B and C, respectively) was incubated without enzyme (control), affinity-purified E. coli RNase E (Rne498) or M. tuberculosis RNase E/G (MycRne), and aliquots withdrawn at times indicated above each lane were analyzed on 20%

Fig. 4 .

 4 Fig. 4. 5´-end dependence of MycRne cleavages. (A) Relative efficiency of MycRne cleavage of fluorescently labelled 5´-phosphorylated and non-phosphorylated oligonucleotides (P-BR13-Fluor and HO-BR13-Fluor, respectively). Each oligonucleotide (~5 pmol) was incubated without (control) or with affinity-purified MycRne) and aliquots withdrawn at times indicated above each lane were analyzed on a 15% (w/v) sequencing-type gel. The signals corresponding to the fluorescently labeled oligonucleotides and their products of cleavage were visualized using a PhosphorImager (Typhoon 8600, Molecular Dynamics) and further quantified employing IMAGEQUANT software. (B) Graphical representation of uncleaved RNA (%) plotted as a function of time (min) indicates that MycRne cleaves the 5´-phosphorylated substrate faster than the nonphosphorylated one. The graph represents the data from one single experiment, which was repeated twice with similar results.

Fig. 5 .

 5 Fig. 5. Comparative cleavage of A27 and U27 by MycRne. (A) 5´ end labelled A27 (on the left) or U27 (on the right) were incubated without enzyme (control) or with 1.5-0.3 µg of native M. tuberculosis RNase E/G (MycRne), and aliquots withdrawn at times indicated above each lane were analyzed on 15% polyacrylamide / urea gels. The 1-nt ladders (lane S1) were generated by partial digestion of A27 and U27 with S1 nuclease, respectively. Each experiment was repeated at least twice and one representative gel image is shown. (B) Cleavage patterns of U27 generated by of Rne498 and MycRne. 5´ end labelled U27 was incubated without enzyme (control), with E. coli RNase E (Rne498) or with M. tuberculosis RNase E/G (MycRne) and aliquots withdrawn at times indicated above each lane were analyzed on a 15% polyacrylamide/ urea gel. Lane S1, a 1 nt ladder prepared by partial digestion of U27 with S1 nuclease. The coordinates of U 1 and U 8 within U27 are indicated. 20

Fig. 6 .

 6 Fig. 6. Cleavage patterns of U27A, U27G, U27C and U27ab generated by Rne498 and MycRne. Each 5´ end labelled substrate derived from U27 by replacement of the U at position 14 by an G (U27G), A (U27A), C (U27C) or abasic (U27ab) residue was incubated without enzyme (control), with E. coli RNase E (Rne498) or with M. tuberculosis RNase E/G (MycRne), and aliquots withdrawn at times indicated above each lane were analyzed on 15% polyacrylamide/ urea gels (panels A, B, C and D, respectively). Indicated are the positions of several nucleotides including the substituted base (A, G, C or the abasic residue (X), respectively) as well as site(s) sensitive to MycRne cleavage. Lane S1, 1 nt ladders generated by partial digestion of each oligonucleotide with S1. Similar patterns were obtained for each substrate in three independent experiments. (E) Schematic views of the substrates. The internucleotide bonds with a moderate and high susceptibility to MycRne cleavage are pointed out by open and filled triangles, respectively.

Fig. 7 .

 7 Fig. 7. 9S RNA processing in vitro and in vivo. (A) 5´-end labelled mycobacterial 9S RNA was incubated without enzyme (control), with E. coli RNase E (Rne498) or with M. tuberculosis RNase E/G (MycRne). Aliquots were withdrawn at times indicated above each lane and analyzed on 10% polyacrylamide / urea gels. The position of M. tuberculosis RNase E/G cleavage site ( * ) was determined employing concomitantly run partial alkaline (L) and RNase T 1 (T1) digests of the same transcript shown on the right. The experiment was repeated three times with similar results. (B) Primer extension analysis of total mycobacterial RNA (lane E) was performed using a 5´-[ 32 P]-labelled primer specific for 5S rRNA. The 5´-end of the in vivo processed 5S rRNA was determined by employing a concomitantly run sequencing ladder (lanes C, T, A and G) generated with the same primer (for details, see Materials & Methods). The experiment was repeated twice with similar results. (C) The sequence of M. tuberculosis 5S rRNA (highlighted with grey shading) and its 5´ end flanking region. A long horizontal arrow shows the direction and position of the primer used for primer extension. The position of in vitro (see panel A) and in vivo (panel B) MycRne cleavage sites that were mapped close to the 5´ end of 5S rRNA are indicated by a single upward and two downward arrows, respectively.

5 Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

  5, 12 and 45 krpm) using 150 µl samples at 3 concentrations (16 µM, 8 µM and 1.6 µM) analysed at 292, 286 and 233 nm, respectively;

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

	The	5'-[ 32 P]-labelled	5S	rRNA-specific	oligonucleotide	5SPEM	(5´-CAG
	TATCATCGGCGCTGGC) and total RNA (150 ng) were co-precipitated with ethanol, the
	pellet was air dried and suspended in 5 µl of annealing buffer (75 mM KCl, 67 mM Tris/HCl,
	pH 8.3), heated to 70°C, quenched on ice, mixed with 5 µl of RT buffet (75 mM Tris/HCl, pH
	8.3, 20 mM MgCl 2 , 4 mM DTT, 0.8 mM each dNTP) containing 4 units of AMV reverse
	transcriptase (Promega) and incubated for 30 min at 42°C. The reaction was stopped by
	adding 10 µl of sequencing dye. The sample was denatured at 95°C for 3 min and further
				6

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530 Copyright 2007 Biochemical Society RNase

  E representing its catalytic domain[12]. The assays were carried out with synthetic ribooligonucleotides BR10, 9SA and OmpC, all of which are well-characterized E. coli

	RNase E substrates

To analyze the self-association state of MycRne, sedimentation equilibrium experiments were performed at different concentrations (1.6 µM, 8 µM, 16 µM, 42 µM and 84 µM) and several speeds. The absorbance scans (fitted simultaneously on single species or self-association models) indicated an apparent molecular mass of 71,100 Da for the monomer. The records at 16 µM and 42 µM fitted best on a monomer-dimer equilibrium (1502 points/concentration, variance 0.000163) with a K D ≈1.8 µM, while the profiles at 84 µM could best be modelled as dimer-tetramer equilibrium (862 points, variance 0.000277) with a K D ≈95 µM. According to these data, the molecular species of MycRne appear mainly as monomers below concentrations of ca. 1 µM, predominantly as dimers up to ca. 100 µM and as tetramers in more concentrated solutions.

Cleavage of oligonucleotide substrates

To test whether MycRne has an endoribonucleolytic activity and to compare its specificity with that of E. coli RNase E, we performed cleavage assays using affinity-purified MycRne and Rne498 (E. coli RNase E; residues 1-498), a C-terminally truncated form of E. coli 8

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

	3 and
	-2, respectively. In other words, strong enhancing effects of nucleotides at certain positions of
	different cleavage sites can potentially neutralize / override negative effects of other sequence
	determinants, resulting in approximately equal efficiency of cleavage.

Like in the case of E. coli RNase E

[35,[START_REF] Walsh | Cleavage of poly(A) tails on the 3´-end of RNA by ribonuclease E of Escherichia coli[END_REF]

, the efficiency of MycRne cleavage of U27 and its mutant variants decreases with the number of the remaining nucleotides that are 10

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530

	nucleotides [35, 46, 47], MycRne apparently prefers to cleave sequences enriched mainly in U
	(but not A) nucleotides. Likewise, the C in U27C differentially affects cleavages by E. coli
	and M. tuberculosis RNase E/G homologues. When placed in certain locations close to the
	scissile bond, the cytosine not only inhibits but also enhances the efficiency of E. coli RNase
	E cleavages ([35], Fig. 6C), whereas, in the case of M. tuberculosis RNase E/G, it only
	inhibits cleavage at nearly every position. This study and others [19,32] demonstrate that the

nucleotides within E. coli RNase E and MycRne cleavage sites. Both endoribonucleases cleave more efficiently in the presence of a G nucleotide upstream of and in close proximity to the point of cleavage. However, unlike E. coli RNase E sites that are usually rich in A/U substrate specificity of RNase E/G endoribonucleases can significantly vary and cannot be easily inferred from previous data that were obtained for a particular RNase E/G homologue. 12

  . The N-terminal domain of the rne gene product has RNase E activity and is non-overlapping with the arginine-rich RNAbinding site. J. Mol. Biol. 255, 349-355. 13 Vanzo, N.F., Li, Y.S., Py, B., Blum, E., Higgins, C.F., Raynal, L.C., Krisch, H.M., and Carpousis, A.J. (1998). Ribonuclease E organizes the protein interactions in the

		Escherichia coli RNA degradosome. Genes Dev. 12, 2770-2781.
	14	Marcaida, M.J., Depristo, M.A., Chandran, V., Carpousis, A.J., and Luisi, B.F. (2006).
		The RNA degradosome: life in the fast lane of adaptive molecular evolution. Trends
		Biochem. Sci. 31, 359-365.
	15	Wachi, M., Umitsuki, G., Shimizu, M., Takada, A., and Nagai, K. (1999). Escherichia
		coli cafA gene encodes a novel RNase, designated as RNase G, involved in processing
		of the 5' end of 16S rRNA. Biochem. Biophys Res. Commun. 259, 483-488.
	16	Li, Z., Pandit, S., and Deutscher, M.P. (1999). RNase G (CafA protein) and RNase E
		are both required for the 5´ maturation of 16S ribosomal RNA. EMBO J. 18, 2878-
		2885.
	17 Okada, Y.18 Okada, Y., Shibata, T., and Matsuhashi, M. (1993). Possible function of the
		cytoplasmic axial filaments in chromosomal segregation and cellular division of
		Escherichia coli. Sci. Prog. 77, 253-264.
	19	Tock, M.R., Walsh, A.P., Carroll, G., and McDowall, K.J. (2000). The CafA protein
		required for the 5'-maturation of 16 S rRNA is a 5'-end-dependent ribonuclease that
		has context-dependent broad sequence specificity. J. Biol. Chem. 275, 8726-8732.
	20	McDowall, K.J., Hernandez, R.G., Lin-Chao, S., and Cohen, S.N. (1993). The ams-1
		and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity
		to each other and cause substitutions within a domain that resembles a product of the
		Escherichia coli mre locus. J. Bacteriol. 175, 4245-4249.
	21	Carpousis, A.J. (2002). The Escherichia coli RNA degradosome: structure, function
		and relationship in other ribonucleolytic multienzyme complexes. Biochem. Soc.
		Trans. 30, 150-155.
	22	Condon, C., and Putzer, H. (2002). The phylogenetic distribution of bacterial
		ribonucleases. Nucl. Acids Res. 30, 5339-5346.

, Wachi, M., Hirata, A., Suzuki, K., Nagai, K., and Matsuhashi, M. (1994).

Cytoplasmic axial filaments in Escherichia coli cells: possible function in the mechanism of chromosome segregation and cell division. J. Bacteriol. 176, 917-922.

Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530 Copyright 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank Dr. Denise Barlow for critically reading the manuscript. This work was supported by grant no. F1707 from the Austrian Science Foundation (V.R.K.), by CNRS (T.R. and T.B.), and by OTKA 034820 from the Hungarian Scientific Research Fund (A.M.). A.Cs. was a recipient of the FEMS and Hungarian State Eötvös fellowships. 13 Biochemical Journal Immediate Publication. Published on 4 Jan 2007 as manuscript BJ20061530 Copyright 2007 Biochemical Society