Amino acid-dependent transfer RNA affinity in a class I aminoacyl-tRNA synthetase

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.     

Active-site assembly in glutaminyl-tRNA synthetase by tRNA-mediated induced fit

Structure-based mutational analysis was employed to probe an unusual intramolecular interaction between partially buried glutamate residues adjacent to the active site of Escherichia coli glutaminyl-tRNA synthetase (GlnRS). The crystal structures of unliganded GlnRS and the GlnRS-tRNA(Gln) complex reveal that the Glu34 and Glu73 side chain carboxylates contact each other only in the tRNA-bound state and that the interaction is formed via mutual induced-fit transitions that occur en route to the ground-state Michaelis complex. Steady-state and transient kinetic analysis of mutant enzymes suggest that the formation of this intermolecular contact is a key event that facilitates the proper formation of the active site. Mutants at both positions destabilize the binding of the substrate glutamine at the opposite side of the active-site cleft, whereas Glu73 appears to play an additional important role by promoting the correct binding of the 3'-acceptor end of tRNA adjacent to both ATP and glutamine. The data suggest the existence of multiple structural pathways by which the binding of tRNA propagates conformational transitions leading to the proper formation of the glutamine binding site. The single-turnover kinetic analysis also establishes that the Glu34 carboxylate does not play a direct enzymatic role as a catalytic base to help deprotonate the tRNA-A76 nucleophilic 2'-hydroxyl group. The elimination of this previously proposed mechanism, together with recent chemical modification experiments in the histidyl-tRNA synthetase system, emphasizes that substrate-assisted catalysis by the phosphate of the aminoacyl adenylate may be a common means by which all tRNA synthetases facilitate the aminoacyl transfer step of the reaction.     

tRNA-dependent active site assembly in a class I aminoacyl-tRNA synthetase

The crystal structure of ligand-free E. coli glutaminyl-tRNA synthetase (GlnRS) at 2.4 A resolution shows that substrate binding is essential to construction of a catalytically proficient active site. tRNA binding generates structural changes throughout the enzyme, repositioning key active site peptides that bind glutamine and ATP. The structure gives insight into longstanding questions regarding the tRNA dependence of glutaminyl adenylate formation, the coupling of amino acid and tRNA selectivities, and the roles of specific pathways for transmission of tRNA binding signals to the active site. Comparative analysis of the unliganded and tRNA-bound structures shows, in detail, how flexibility is built into the enzyme architecture and suggests that the induced-fit transitions are a key underlying determinant of both amino acid and tRNA specificity.     

Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase

Glutamyl-tRNA synthetase (GluRS) is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for specific amino acid recognition and activation. We analyzed the role of tRNA in amino acid recognition by crystallography. In the GluRS*tRNA(Glu)*Glu structure, GluRS and tRNA(Glu) collaborate to form a highly complementary L-glutamate-binding site. This collaborative site is functional, as it is formed in the same manner in pretransition-state mimic, GluRS*tRNA(Glu)*ATP*Eol (a glutamate analog), and posttransition-state mimic, GluRS*tRNA(Glu)*ESA (a glutamyl-adenylate analog) structures. In contrast, in the GluRS*Glu structure, only GluRS forms the amino acid-binding site, which is defective and accounts for the binding of incorrect amino acids, such as D-glutamate and L-glutamine. Therefore, tRNA(Glu) is essential for formation of the completely functional binding site for L-glutamate. These structures, together with our previously described structures, reveal that tRNA plays a crucial role in accurate positioning of both L-glutamate and ATP, thus driving the amino acid activation.     

Synthesis of beta-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases

The aminoacyl-beta-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized glutamyl-beta-ketophosphonate-adenosine (Glu-KPA) and glutaminyl-beta-ketophosphonate-adenosine (Gln-KPA), and have tested them as inhibitors of their cognate aaRS, and of a non-cognate aaRS. Glu-KPA is a competitive inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS) with a K(i) of 18microM with respect to its substrate glutamate, and binds at one site on this monomeric enzyme; the non-cognate Gln-KPA also binds this GluRS at one site, but is a much weaker (K(i)=2.9mM) competitive inhibitor. By contrast, Gln-KPA inhibits E. coli glutaminyl-tRNA synthetase (GlnRS) by binding competitively but weakly at two distinct sites on this enzyme (average K(i) of 0.65mM); the non-cognate Glu-KPA shows one-site weak (K(i)=2.8mM) competitive inhibition of GlnRS. These kinetic results indicate that the glutamine and the AMP modules of Gln-KPA, connected by the beta-ketophosphonate linker, cannot bind GlnRS simultaneously, and that one Gln-KPA molecule binds the AMP-binding site of GlnRS through its AMP module, whereas another Gln-KPA molecule binds the glutamine-binding site through its glutamine module. This model suggests that similar structural constraints could affect the binding of Glu-KPA to the active site of mammalian cytoplasmic GluRSs, which are evolutionarily much closer to bacterial GlnRS than to bacterial GluRS. This possibility was confirmed by the fact that Glu-KPA inhibits bovine liver GluRS 145-fold less efficiently than E. coli GluRS by competitive weak binding at two distinct sites (average K(i)=2.6mM). Moreover, these kinetic differences reveal that the active sites of bacterial GluRSs and mammalian cytoplasmic GluRSs have substantial structural differences that could be further exploited for the design of better inhibitors specific for bacterial GluRSs, promising targets for antimicrobial therapy.     

A fluorescence spectroscopic study of glutaminyl-tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism

Interaction between Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNA(Gln) and ATP bind with dissociation constants of 460, 0.22 and 180 microM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme-bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5'-(methyl)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive-ligand binding. tRNA also only has a weak effect on phosphodiester binding. Selectively pyrene-labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNA(Gln) crystal structure indicates that no major change in shape and size occurs upon tRNA(Gln) binding to GlnRS. 5,5'-Bis(8-anilino-1-naphthalene sulfonate) (bis-ANS), a non-covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNA(Gln) binding. We conclude that lack of tRNA-independent pyrophosphate-exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.     

Activation of methionine by Escherichia coli methionyl-tRNA synthetase

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene Descent, Duplication, and Horizontal Transfer in the Evolution of Glutamyl- and Glutaminyl-tRNA Synthetases

In translation, separate aminoacyl-tRNA synthetases attach the 20 different amino acids to their cognate tRNAs, with the exception of glutamine. Eukaryotes and some bacteria employ a specific glutaminyl-tRNA synthetase (GlnRS) which other Bacteria, the Archaea (archaebacteria), and organelles apparently lack. Instead, tRNA^Gln is initially acylated with glutamate by glutamyl-tRNA synthetase (GluRS), then the glutamate moiety is transamidated to glutamine. Lamour et al. [(1994) Proc Natl Acad Sci USA 91:8670–8674] suggested that an early duplication of the GluRS gene in eukaryotes gave rise to the gene for GlnRS—a copy of which was subsequently transferred to proteobacteria. However, questions remain about the occurrence of GlnRS genes among the Eucarya (eukaryotes) outside of the ``crown' taxa (animals, fungi, and plants), the distribution of GlnRS genes in the Bacteria, and their evolutionary relationships to genes from the Archaea. Here, we show that GlnRS occurs in the most deeply branching eukaryotes and that putative GluRS genes from the Archaea are more closely related to GlnRS and GluRS genes of the Eucarya than to those of Bacteria. There is still no evidence for the existence of GlnRS in the Archaea. We propose that the last common ancestor to contemporary cells, or cenancestor, used transamidation to synthesize Gln-tRNA^Gln and that both the Bacteria and the Archaea retained this pathway, while eukaryotes developed a specific GlnRS gene through the duplication of an existing GluRS gene. In the Bacteria, GlnRS genes have been identified in a total of 10 species from three highly diverse taxonomic groups: Thermus/Deinococcus, Proteobacteria γ/β subdivision, and Bacteroides/Cytophaga/Flexibacter. Although all bacterial GlnRS form a monophyletic group, the broad phyletic distribution of this tRNA synthetase suggests that multiple gene transfers from eukaryotes to bacteria occurred shortly after the Archaea–eukaryote divergence.     

The 2 A crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue

Leucyl-, isoleucyl- and valyl-tRNA synthetases are closely related large monomeric class I synthetases. Each contains a homologous insertion domain of approximately 200 residues, which is thought to permit them to hydrolyse ('edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid. We describe the first crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A resolution. The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure. This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional flexibly inserted domain. Comparison of native enzyme and complexes with leucine and a leucyl- adenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes significant conformational changes in the active site required for amino acid activation and tight binding of the adenylate. These changes are propagated to more distant regions of the enzyme, leading to a significantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.     

ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding

Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.     

tRNA-dependent aminoacyl-adenylate hydrolysis by a nonediting class I aminoacyl-tRNA synthetase

Glutaminyl-tRNA synthetase generates Gln-tRNA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln) and requires tRNA to synthesize the activated aminoacyl adenylate in the first step of the reaction. To examine the role of tRNA in amino acid activation more closely, several assays employing a tRNA analog in which the 2'-OH group at the 3'-terminal A76 nucleotide is replaced with hydrogen (tRNA(2'HGln)) were developed. These experiments revealed a 10(4)-fold reduction in kcat/Km in the presence of the analog, suggesting a direct catalytic role for tRNA in the activation reaction. The catalytic importance of the A76 2'-OH group in aminoacylation mirrors a similar role for this moiety that has recently been demonstrated during peptidyl transfer on the ribosome. Unexpectedly, tracking of Gln-AMP formation utilizing an alpha-32P-labeled ATP substrate in the presence of tRNA(2'HGln) showed that AMP accumulates 5-fold more rapidly than Gln-AMP. A cold-trapping experiment revealed that the nonenzymatic rate of Gln-AMP hydrolysis is too slow to account for the rapid AMP formation; hence, the hydrolysis of Gln-AMP to form glutamine and AMP must be directly catalyzed by the GlnRS x tRNA(2'HGln) complex. This hydrolysis of glutaminyl adenylate represents a novel reaction that is directly analogous to the pre-transfer editing hydrolysis of noncognate aminoacyl adenylates by editing synthetases such as isoleucyl-tRNA synthetase. Because glutaminyl-tRNA synthetase does not possess a spatially separate editing domain, these data demonstrate that a pre-transfer editing-like reaction can occur within the synthetic site of a class I tRNA synthetase.     

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.     

Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.     

The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.     

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.     

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.     

Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA(Gln) interaction.

This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA(Gln). Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA(Gln).GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA(Gln) species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3'-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA(Gln) are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.