(0)388417041, Fax: +33(0)388602218, Email: H.Becker/at/ibmc.u-strasbg.fr Correspondence may also be addressed to Daniel Kern. +33(0)388417092 +33(0)388602218; Email: D.Kern/at/ibmc.u-strasbg.fr
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Flawless protein translation requires a full set of 20 types of perfectly paired aminoacyl-tRNAs. It was de facto expected that each cell should possess a full set of 20 aminoacyl-tRNA synthetases capable of matching each of the 20 natural amino acids to the cognate tRNAs (1). Recent studies on synthesis of glutaminyl-tRNAGln have forced a revision of this assumption (2). This aminoacylated tRNA displays the unique feature of being formed by kingdom-specific pathways (3,4). Eukaryotes and a small subset of bacteria use the common route of tRNA aminoacylation, whereby the GlnRSs attach glutamine directly onto tRNAGln (5). The majority of bacteria and all archaea use an indirect pathway involving a tRNA-dependent amidotransferase that compensates for the absence of GlnRS (3,4,6,7). In this alternate route, glutamyl-tRNAGln, a misacylated species formed by a non-discriminating GluRS (8), is amidated by a domain-specific AdT thereby generating the correctly paired glutaminyl-tRNAGln (3,4). In bacteria, AdTs are composed of three subunits, GatC, GatA and GatB assembled in a heterotrimeric enzyme called GatCAB (4,9,10). While half of the archaea possess this heterotrimeric AdT, they all possess an archaeal-specific AdT called GatDE (3,9–11). Transamidation-catalyzed formation of Gln-tRNAGln is so widespread among prokaryotes that GlnRS is rarely found in bacteria and has so far never been identified in archaea. Deinococcus radiodurans is one of those rare bacteria possessing a GlnRS (12,13).
All aaRSs are assembled piece-wise and often display domains appended to the two ubiquitous functional modules that are the catalytic and anticodon-binding domains (14–18). However, all bacterial GlnRSs studied so far lack any appended domain. The presence in D. radiodurans GlnRS of 220 additional amino acids drew our attention. This extension is located in the C-terminal part of the protein and constitutes an appendix of its anticodon-binding domain (12,19,20). Sequence comparison shows that the C-terminal half of this appendix is homologous to a family of bacterial and yeast proteins of unknown function named Yqey (PFAM id: PF02637, 21). There are ~100 known Yqey proteins that on average are 150 amino-acid-long, and interestingly, share sequence homologies with the C-terminal domain of GatB and GatE, the tRNA-binding subunits of bacterial and archaeal AdTs (22,23). The presence of the gatB or gatE gene signals the presence of an AdT in an organism since the GatB and GatE subunits are exclusively found in association with the other subunits constituting the AdTs and, except the Yqey proteins, have no homologs. Among these proteins, the one encoded by D. radiodurans is unique (12). While in all organisms, when present, Yqey is encoded in an autonomous ORF, in D. radiodurans its gene is fused in frame downstream that of GlnRS. As a result, Dr GlnRS is formed by the fusion of structural protein modules involved in the direct and indirect pathways of glutaminyl-tRNAGln formation. In order to gain insight into the function of the Yqey domain appended to Dr GlnRS, functional and structural investigations were undertaken with this atypical aaRS. Remarkably, the Yqey domain has a disordered orientation in the structure of GlnRS where it acts as a tRNAGln affinity enhancer. Mechanistic implications are discussed.
Complexes between GlnRS and Yqey and their variants and tRNAGln were visualized by band-shift after PAGE fractionation and quantified. For that, constant amounts of tRNAGln transcript (5×105cpm) were incubated with increasing amounts of FL-GlnRS, -GlnRS, FL-Yqey or -Yqey in the presence of 10% glycerol, 12mM MgCl2 and 30mM KCl during 20min at room temperature. Some assays contained 50nM of Neisseria meningitidis tRNAAsp transcript. Ten-microliter samples were analyzed by native PAGE (8%) in 89mM Tris-Borate pH 8.3at 4°C. Free and protein-bound [32P] labeled tRNAs were revealed by scanning the image plates after 12h exposure with a Fuji Bioimager and quantified using the volume rectangle tool of the Quantify One Software (BIO-RAD).
For tRNA aminoacylation, 25µl reaction mixtures contained 100mM Na-HEPES pH 7.2, 2mM ATP, 10mM MgCl2, 30mM KCl, 0.5mg·ml−1 bovine serum albumin, 50µM l-[14C]Gln (351cpm·pmol−1), 1–2mg·ml−1 D. radiodurans unfractionated tRNA (Gln charging:0.8nmol·mg−1) or 1–2μM tRNAGln(UUG) or tRNAGln(CUG) transcripts (Gln charging:25nmol·mg−1) and appropriate amounts of FL- or Δ-GlnRS for initial rate or plateau measurements. For Gln KM determinations, reaction mixtures contained 10–100µM l-[14C]Gln and a saturating concentration of tRNA; for tRNA KM determinations they contained 5μM l-[3H]Gln (560cpm·pmol−1) and 1.74–10.9 or 10–111nM tRNAGln in the presence of FL- and Δ-GlnRS, respectively or either 0.2–1μM or 2–75μM tRNAGln(UUG) transcript. The amount of [3H] or [14C]glutaminyl-tRNA formed after incubations from 30s to 30min at 37°C was determined in 10-μl aliquots (29).
Structures of Ec GlnRS, either in the free state (PDB id: 1NYL) or in complex with ligands (tRNA, AMP, Gln; PDB id: 1O0B) were used for comparison with that of Dr GlnRS using LSQMAN (34). Superimposition was performed with a distance cutoff of 3.5Å. Root-mean-square deviation (RMSD) values determined for the whole GlnRS-core or individual domains are based on equivalent main chain atom positions (N, Cα, C). This structural superimposition allowed annotation of the alignment.
A normal mode analysis of the structure of Dr GlnRS based on the elastic network model was performed using the ElNemo web service (34,35). Sets of normal mode perturbed models were generated for the first five low frequency modes. The model showing the lowest RMSD with Ec GlnRS complexed with the ligands obtained by combining mode 7 and 9 was used for tRNA docking. A series of models, intermediate between the latter and the X-ray structure, were used to predict a plausible conformational pathway between the free and tRNA-bound states. The regions of the enzyme that are not observed in the electron density map and, thus are absent in the current structure, were modeled using Modeller (36). The C-terminal domain was derived from alignment of the Yqey domains and the structure of the Yqey protein of Bacillus subtilis (PDB id: 1NG6).
Next, the C-terminal appendix of Dr GlnRS was aligned with all known Yqey sequences, including that of B. subtilis, and with those of bacterial GatB and GatE proteins (Figure 1C). This alignment shows a moderate degree of sequence similarity, since out of the 215 amino acids constituting the extension of Dr GlnRS, only the 150 most distal align with other Yqey proteins, with highest similarity in the 30 last residues (Figure 1C).
The relatively weak sequence homology between the Yqey domain of Dr GlnRS and GatB subunits raises the question of their conformational homology. A first answer came from an immunological approach. Western blots (Figure 2A) show that Dr GlnRS is recognized by anti-AdT antibodies (lane 2) specific to the GatA and GatB subunits of T. thermophilus AdT (lane 1). These antibodies do not recognize T. thermophilus GlnRS (Figure 2A, lane 3) lacking Yqey (39), suggesting that anti-GatB antibodies recognize the Yqey domain of Dr GlnRS. The unambiguous proof of this assumption came from comparative experiments done with three GlnRS variants, namely a truncated form lacking the entire Yqey domain (Δ-GlnRS), a full length Yqey domain of 220 amino acids (FL-Yqey) and a deletion variant of the extension without the 61 residue-long linker (Δ-Yqey). Δ-GlnRS is no longer recognized by the anti-GatB antibodies, while both FL-Yqey and Δ-Yqey are recognized (Figure 2A, lanes 5–7). Thus, the Yqey domain of Dr GlnRS is solely responsible for the cross-reaction with anti-GatB antibodies. Conversely, antibodies directed against Dr GlnRS, specifically recognize GatB from T. thermophilus AdT and not GatA (Figure 2A, lanes 8 and 9). Additionally, these antibodies are unable to recognize T. thermophilus GlnRS indicating the absence of common epitopes in the core of both enzymes (Figure 2A, lane 10). Altogether this demonstrates a structural resemblance between the Yqey domain of Dr GlnRS and the family of Yqey proteins. In other words, it confirms the chimerical nature of Dr GlnRS suspected from sequence analysis.
As could be anticipated from sequence conservation and straightforward molecular replacement, the GlnRS-core is similar to that of E. coli (Figure 3A). The structures can be superimposed with a RMSD of 1.9–2.3Å (Supplemental Table 1 for RMSD analysis). Below, the E. coli nomenclature is used to describe the protein which can be divided as follows: an active site domain (ASD) composed of a Rossmann or dinucleotide fold (DNF) and the acceptor-stem binding domain (ABD), an intermediate helical subdomain (HSD) and the anticodon-binding domain (ACD) made of a proximal and a distal β-barrel (PBB, DBB).
Figure 3B highlights the sequence and structure conservations between Ec and Dr GlnRSs. The ASDs of the two proteins are similar and present the HI/LGH and T/MSK signatures of class I synthetases. This part of both GlnRSs is deprived of any insertion–deletion sequence. The unique difference concerns position of a helix in the ABD which varies from more than 3.5Å (depicted in white in Figure 3B). It adopts an open conformation in Dr GlnRS and closes upon tRNA binding in the ligand-complexed Ec GlnRS. Overall, low RMSD values (0.8–1.3Å) are observed in the ASD, as well as in the HSD.
The major structural differences between Ec and Dr GlnRSs occur in their ACDs. The PBB of Dr GlnRS contains two insertions in surface loops that are not seen in the density. The first consists of 27 residues forming an extended loop (535–565) located at the bottom of the catalytic cleft; this element is replaced by a short turn in Ec GlnRS. The second concerns five additional residues in a loop (610–620) opposite to the anticodon-loop binding site. The PBB has been proposed as a module promoting communication between the catalytic center and the DBB where the anticodon of tRNA is bound. Accordingly, this part of the structure shows the highest RMSD deviations with the complexed Ec GlnRS. The DBB of Dr GlnRS also carries other idiosyncrasies, namely an additional element of 34 residues (410–433) that folds in a helix followed by two short β-strands sitting next to the minor groove of the anticodon stem of tRNA and a deletion of 8 residues found in a loop that would be close to nucleotides 34–36 of the tRNA anticodon. This loop (497–505) is well defined in Dr GlnRS whereas it has not been observed in the structure of Ec GlnRS either in free form or complexed with the substrates, probably due to a greater mobility related to its larger size. Overall, this domain appears to be more rigid than the PBB as revealed by RMSD values of 1.2–1.3Å.
The amino acids of Ec GlnRS shown to participate in binding of the substrates are almost fully conserved in Dr GlnRS: all residues involved in recognition of Gln by direct or solvent mediated interactions are present, as well as most residues constituting the ATP pocket. Even more striking is the high level of conservation of the residues which in Ec GlnRS develop specific interactions with tRNA (Figure 1A).
Among the 40 residues of Ec GlnRS which contact tRNA (12 with main-chain atoms and 28 with lateral-chain atoms) 25 are strictly conserved and 9 present structural conservative changes (e.g. Ile/Leu, Glu/Gln) in Dr GlnRS. Interestingly, 4 residues of Dr GlnRS are unable to make the interactions described for the E. coli complex. Among the 10 residues of Ec GlnRS contacting ATP, 8 are conserved or substituted by equivalent residues and the 13 contacting Gln are conserved, except one substituted by a functionally equivalent residue (M of the MSK motif replaced by T) (Supplemental Table 2).
Comparative experiments deciphered the effects of Yqey on the catalytic properties of Dr GlnRS. First Δ-GlnRS, like FL-GlnRS and other GlnRSs, requires cognate tRNA for Gln activation. Further, the kinetic constants of Gln activation and tRNA aminoacylation (Table 2) show that both FL- and Δ-GlnRSs exhibit similar KM's for Gln in ATP–PPi exchange as well as in tRNA charging, suggesting that Yqey is not involved in binding of the amino-acid substrate. This activation is specific since the two enzyme forms do not catalyze Glu-dependent ATP–[32P]PPi exchange under the conditions where Gln promotes the exchange and despite the ability of Ec GlnRS to bind Glu (40). On the other hand, FL- and Δ-GlnRSs catalyze amino-acid activation and tRNA aminoacylation, with similar rate since deletion of the Yqey domain only slightly decreases kcat's of ATP–PPi exchange and tRNA charging (Table 2). This suggests that Yqey does not contain essential residues for catalysis or active conformation of the enzyme. The domain, however, increases the affinity of the enzyme for cognate tRNAGln, as revealed by the significant KM decrease of FL-GlnRS for this substrate, especially pronounced for ATP–PPi exchange. Taken together, the Yqey flexible module increases the catalytic efficiency of GlnRS for both tRNAGln-dependent Gln activation and tRNAGln charging and is involved in binding of tRNAGln to GlnRS.
Next, the capacity of Dr GlnRS to make a functional distinction between its two tRNAGln isoacceptors, tRNAGln(UUG) and tRNAGln(CUG), and the possible role of Yqey in this distinction, were investigated. Thus charging of the two transcripts by FL- and Δ-GlnRSs was tested. In contrast to Ec GlnRS that charges transcript and modified tRNAGln with similar efficiencies (42), Dr GlnRS aminoacylates the tRNAGln(UUG) transcript significantly less efficiently than modified tRNAGln and the two transcripts with distinct rates, the tRNAGln(UUG) transcript being charged one order of magnitude faster than the tRNAGln(CUG) transcript (not shown). Further, Δ-GlnRS charges the tRNAGln(UUG) transcript one order of magnitude slower than FL-GlnRS and is unable to charge the tRNAGln(CUG) transcript to a detectable level (not shown). This indicates that Dr GlnRS can charge the two isoacceptors, although with distinct efficiencies and that Yqey does not support preferential aminoacylation of one of them. Interestingly, the two tRNAs display 73% identity. Sequence analysis shows that among the elements determining glutaminylation of E. coli tRNAGln (43–45), 10 are conserved in D. radiodurans tRNAGln(UUG) (G73, G2–C71, G3–C70, G10, U34, U35 and G36, U38) and 8 in tRNAGln(CUG) (G73, G2–C71, G3–C70, G10, U35, G36, U1–A72). Among the positions involved in E. coli glutaminylation, both tRNAs differ by positions 1–73, 34 and 38 (respectively A1–U73, U34, U38 and U1–A73, C34, C38 in tRNAGln(UUG) and tRNAGln (CUG)).
Interestingly, the above data suggest a role of post-transcriptional modifications in tRNA aminoacylation (Table 2). FL-GlnRS aminoacylates modified tRNAGln(UUG) ~80-fold more efficiently than the transcript as a result of a strong kcat increase and a faint KM decrease. The poor affinity of Δ-GlnRS for the tRNAGln(UUG) transcript did not allow determination of the individual kinetic constants for the unmodified molecule. However, under first-order kinetic conditions with respect to tRNA, the kcat/KM ratio could be measured directly. It indicates that Δ-GlnRS aminoacylates the transcript ~5 order of magnitude less efficiently than FL-GlnRS. Since the absence of Yqey decreases charging efficiency of the transcript much more than that of native tRNAGln (~23 000-fold), participation of the post-transcriptional modifications in the Yqey-dependent formation of the competent GlnRS·tRNAGln complex is not a side effect. It is specific to D. radiodurans tRNAGln since modified E. coli tRNAGln is a poor substrate for Dr GlnRS (not shown). The nature of the peculiar modifications in D. radiodurans tRNAGln awaits to be characterized. Interestingly, it has been shown that in E. coli the post-transcriptionally modified nucleotides of tRNAGln are not involved in glutaminylation (44).
Given this crucial contribution of Yqey in Dr GlnRS for binding tRNAGln, the capacity of FL-Yqey (Figure 4C) and Δ-Yqey domains to bind the tRNAGln(UUG) transcript was checked. Both Yqey variants are unable to shift the cognate tRNA transcript, even at very high protein concentrations. Thus, isolated Yqey is not a tRNA binder per se. Furthermore this domain is unable to restore in trans (i.e. by addition of increasing amounts of FL- or Δ-Yqey domains), a tRNAGln-binding capability to Δ-GlnRS (Figure 4C).
The present study establishes that the Yqey domain of Dr GlnRS is an actor in tRNA aminoacylation and suggests that it contributes to tRNA binding. More precisely, kinetic data unambiguously show that Yqey is a tRNA affinity enhancer for Dr GlnRS. Yqey must be covalently attached to the GlnRS core to fullfil its role, since it is unable to act in trans when added in free form to the truncated enzyme. Its connection through a long polypeptide linker to GlnRS constitutes another peculiarity distinguishing Yqey from other aaRS-appended modules. This feature confers unprecedented properties to Dr GlnRS.
Formation of the catalytically competent GlnRS·tRNAGln complex triggered by Yqey is tuned by the modified nucleotides of tRNA, as supported by kinetic data. Although the details of this tuning remain to be deciphered, it appears that the modifications contribute more to the formation of a competent complex than to the primary binding of tRNA. The way whereby Yqey enhances affinity of Dr GlnRS for native post-transcriptionally modified tRNAGln is intriguing since the core enzyme conserves most of the residues of Ec GlnRS involved in tRNA recognition, and thus is expected to be fully competent to charge efficiently tRNAGln. Because appended Yqey increases kcat of tRNA charging and decreases KM, it may act by two ways, either in favoring formation of the competent complex by distorting and reorienting the partners to a productive state or in increasing affinity of GlnRS for tRNAGln by enhancing the rate of complex formation and decreasing that of its dissociation.
Finally, from the viewpoint of prokaryote genomes, Yqey genes are often located in an operon (Supplemental Table 3). The gene of ribosomal S21 protein is mostly found associated with that of Yqey, but other genes can also be found, although less frequently. The grouping of Yqey and S21 genes in an operon is independent of the presence or absence of AdT and when present, independent of its activity, either Glu- or Asp-AdT. Protein S21 is peculiar to eubacteria and S21-deficient 30S ribosomes are unable to bind mRNA and to initiate protein synthesis in E. coli (51). Assuming that Yqey-containing operons encode proteins of related functions, Yqey may be a cofactor involved in protein synthesis.
In a structural perspective, and at first glance, the insertions observed in the core enzyme cannot easily account for the observations. However, although crystallography does not yet answer unambiguously how the extension participates in the aminoacylation function of Dr GlnRS, it does not exclude a direct contact of the Yqey domain with tRNA. This domain contains helical subdomains that may allow such contacts with tRNA helices, as found in the yeast AspRS extension (52), but their content of basic residues is poor, except for the most proximal helix to the GlnRS core (Figure 1). The linker making the connection with this core is long enough to allow Yqey to contact any region of the tRNA, even its acceptor or anticodon ends (Figure 5). If so, the extension would clamp the tRNA on the GlnRS core in position optimal for activity. Reminiscent to what occurs in the Ec GlnRS·tRNAGln complex, where base-pair U1–A72 opens prior entry of the tRNAGln-acceptor end in the catalytic site (30), and in AdT-systems where this pair is a recognition determinant for amidation of tRNA-bound Glu or Asp into Gln or Asn (23,53), Yqey in Dr GlnRS may trigger the formation of the competent complex by contacting the U1–A72 pair of tRNAGln. Additional structural data are awaited to test this hypothesis and to decipher the puzzling tRNA recognition by Dr GlnRS. However, the presence of Yqey in free form regardless whether GlnRS is present or absent in the organism such as in B. subtilis deprived of GlnRS and which uses the indirect pathway for Gln-tRNAGln formation (4), suggests that this domain may exert additional functions not directly related to Gln-tRNAGln formation, like stabilization of the aa-tRNA, regulation of its synthesis, or coupling of its synthesis with other metabolic pathways. Alternatively, it cannot be excluded that even in free form Yqey participates in cellular functions involving protein–nucleic acids interactions such as binding to an aaRS·tRNA complex to increase efficiency and/or specificity of tRNA aminoacylation. In this case, the protein's properties would resemble Ybak (54,55) which binds Cys-tRNAPro complexed with ProRS and promotes hydrolysis of the mischarged tRNA. However, it does so by acting exclusively in trans, in contrast to the role of Yqey in Dr GlnRS, which can be performed only when it is present in cis.
Considerations about evolution of tRNA glutaminylation and asparaginylation led to the proposal that the transamidation pathway constitutes the primitive route to Gln-tRNAGln and Asn-tRNAAsn formation (9,10). These routes preceded the emergence of the modern GlnRS and AsnRS, able to synthesize their corresponding aa-tRNAs by charging preformed Gln and Asn onto their cognate tRNAs (9,10). Thus, by amidating Glu and Asp charged on tRNAGln and tRNAAsn, AdT was probably the first enzyme able to form Asn and Gln for protein synthesis purposes. Sequence comparisons and phylogenetic trees revealed that GlnRS evolved from a duplicated gene of GluRS in eukaryotes, and was transferred in various eubacterial species by lateral gene transfer (5). In D. radiodurans, Gln-tRNAGln is formed by the modern GlnRS whereas Asn-tRNAAsn is formed by the indirect pathway involving AdT (13). In this organism recruitment, by GlnRS, of a domain belonging to the AdT, a partner of the ancient pathway of Gln-tRNAGln and Asn-tRNAAsn synthesis is surprising, and reveals an unexpected interrelation between the ancient and the modern pathways of amide aa-tRNA formation. Since the Yqey domain is only exceptionally appended to bacterial GlnRS, fusion of both occurred late in evolution and was probably related to acquisition of peculiar properties of GlnRS. Investigations are underway to identify the precise functions of the free and GlnRS-bound Yqey.
This work was supported by the Université Louis Pasteur (Strasbourg), Centre National de la Recherche Scientifique (CNRS), and grants from Association pour la Recherche sur le Cancer (ARC) and ACI Biologie Cellulaire, Moléculaire et Structurale. The authors acknowledge the team of ID14 beamlines at ESRF for assistance during data collection. M.D. benefitted from FEBS and CNRS grants and C.S. was the recipient of a Marie Curie European Reintegration Grant (MERG-CT-2004-004898). Funding to pay the Open Access publication charge was provided by the CNRS.
Conflict of interest statement. None declared.