Covalent methionylation of Escherichia coli methionyl-tRNA synthethase: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [32P]PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.     

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.     

Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis

Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.     

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).     

Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes

Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.     

Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R: partial purification and properties of methionyl-transfer ribonucleic acid synthetase and methionyl-transfer ribonucleic acid formyltransferase

The initiation of protein synthesis by Streptococcus faecalis R grown in folate-free culture occurs without N-formylation or N-acylation of methionyl-tRNA(f) (Met). Methionyl-tRNA synthetase and methionyl-tRNA formyltransferase were partially purified from S. faecalis grown under normal culture conditions in the presence of folate (plus-folate); the general properties of the enzymes were determined and compared with the properties of the enzymes purified from wild-type cells grown in the absence of folate (minus-folate). S. faecalis methionyl-tRNA synthetase displays optimal activity at pH values between 7.2 and 7.8, requires Mg(2+), and has an apparent molecular weight of 106,000, as determined by gel filtration, and 127,000, as determined by sucrose density gradient centrifugation. The K(m) values of plus-folate methionyl-tRNA synthetase for each of the three substrates in the aminoacylation reaction (l-methionine, adenosine triphosphate, and tRNA) are nearly identical to the respective substrate Michaelis constants of minus-folate methionyl-tRNA synthetase. Furthermore, both plus- and minus-folate S. faecalis methionyl-tRNA synthetases catalyze, at equal rates, the aminoacylation of tRNA(f) (Met) and tRNA(m) (Met) isolated from either plus-folate or minus-folate cells. S. faecalis methionyl-tRNA formyltransferase displays optimal activity at pH values near 7.0, is stimulated by Mg(2+), and has an apparent molecular weight of approximately 29,900 when estimated by sucrose density gradient centrifugation. The K(m) value of plus-folate formyltransferase for plus-folate Met-tRNA(f) (Met) does not differ significantly from that of minus-folate formyltransferase for minus-folate Met-tRNA(f) (Met). Both enzymes can utilize either 10-formyltetrahydrofolate or 10-formyltetrahydropteroyltriglutamate as the formyl donor; the Michaelis constant for the monoglutamyl pteroyl coenzyme is slightly less than that of the triglutamyl pteroyl coenzyme for both transformylases. Tetrahydrofolate and uncharged tRNA(f) (Met) are competitive inhibitors of both plus- and minus-folate S. faecalis formyltransferase; folic acid, pteroic acid, aminopterin, and Met-tRNA(m) (Met) are not inhibitory. These results indicate that the presence or absence of folic acid in the culture medium of S. faecalis has no apparent effect on either methionyl-tRNA synthetase or methionyl-tRNA formyltransferase, the two enzymes directly involved in the formation of formylmethionyl-tRNA(f) (Met). Therefóre, the lack of N-formylation of Met-tRNA(f) (Met) in minus-folate S. faecalis is due to the absence of the formyl donor, a 10-formyl-tetrahydropteroyl derivative. Although the general properties of S. faecalis methionyl-tRNA synthetase are similar to those of other aminoacyl-tRNA synthetases, S. faecalis methionyl-tRNA formyltransferase differs from other previously described transformylases in certain kinetic parameters.     

Specific hydrolysis of methionyl-tRNA Met f catalyzed by a purified peptide.

A peptide initiation factor purified from rat liver and promoting the binding of initiator tRNA and model initiators to 40S and 80S ribosome at an acid pH liberates methionine and N-acetylmethionine from Trna Met f at neutral reaction. Phenylalanyl-tRNA, N-acetylphenylalanyl-tRNA and methionyl-tRNA Met m are not hydrolyzed under the same conditions. Hydrolysis of methionyl-tRNA Met f is stimulated by the presence of the 40S ribosomal subunit and preceeds at 37 degrees C until all the substrate has been split. No hydrolysis of initiator tRNA or N-acetylmethionyl-tRNA Met f occurs at 0 degrees C. Hydrolysis is slightly stimulated by GTP and MG2+ but not by KCl. The binding and hydrolyzing activity associated with a single protein factor may have an important function in regulating the rate of peptide initiation.     

The appended C-domain of human methionyl-tRNA synthetase has a tRNA-sequestering function.

An ancillary RNA-binding domain is appended to the C-terminus of human methionyl-tRNA synthetase. It comprises a helix-turn-helix (HTH) motif related to the repeated units of the linker region of bifunctional glutamyl-prolyl-tRNA synthetase, and a specific C-terminal KGKKKK lysine-rich cluster (LRC). Here we show by gel retardation and tRNA aminoacylation experiments that these two regions are important for tRNA binding. However, the two pieces of this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endowed with a tRNA-sequestering activity and confers on MetRS a rate-limiting dissociation of aminoacylated tRNA. Elongation factor EF-1alpha enhanced the turnover in the aminoacylation reaction. In contrast, the LRC region is most probably involved in accelerating the association step of deacylated tRNA. These two nonredundant RNA-binding motifs strengthen tRNA binding by the synthetase. The native form of MetRS, containing the C-terminal RNA-binding domain, behaves as a processive enzyme; release of the reaction product is not spontaneous, but may be synchronized with the subsequent step of the tRNA cycle through EF-1alpha-assisted dissociation of Met-tRNA(Met). Therefore, the eukaryotic-specific C-domain of human MetRS may have a dual function. It may ensure an efficient capture of tRNA(Met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instigate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1alpha.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from Escherichia coli

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA_s^Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k_R, and for the dissociation, k_D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k_R values of 250 μm^?1 s^?1 reflect similar behaviour for antico-operative binding of both tRNA_s^Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA_s^Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA^Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.     

Purification and NMR studies of [methyl-13C]methionine-labeled truncated methionyl-tRNA synthetase

A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed. With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase [Barker, D.G., Ebel, J.-P., Jakes, R., & Bruton, C.J. (1982) Eur. J. Biochem. 127, 449], can be obtained. The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases. A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies. With this procedure, truncated methionyl-tRNA synthetase labeled with [methyl-13C]methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase. Both carbon-13 and proton observe-heteronuclear detect NMR experiments were used to observe the 13C-enriched methyl resonances of the 17 methionine residues in the truncated synthetase. In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR. Titration of the synthetase, monitoring the chemical shifts of resonances B and M (Figure 3), with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry

Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the -NH₂ group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 ± 0.1 and 4.3 ± 0.1 mol of [¹⁴C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.     

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation

The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.     

Affinity labeling of aminoacyl-tRNA synthetases with adenosine triphosphopyridoxal: probing the Lys-Met-Ser-Lys-Ser signature sequence as the ATP-binding site in Escherichia coli methionyl-and valyl-tRNA synthetases

Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes [Tagaya, M., & Fukui, T. (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T. (1988) FEBS Lett. 229, 261-264], was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS). Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities. Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS. ATP or MgATP protected the enzyme from inactivation. The labeling with AP3-PL was also applied to E. coli valyl-tRNA synthetase (ValRS). Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS. AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS. In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909. We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP. AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of analogues of methionine and methionyl adenylate to sample conformational changes during catalysis in Escherichia coli methionyl-tRNA synthetase

Binding of methionine to methionyl-tRNA synthetase (MetRS) is known to promote conformational changes within the active site. However, the contribution of these rearrangements to enzyme catalysis is not fully understood. In this study, several methionine and methionyl adenylate analogues were diffused into crystals of the monomeric form of Escherichia coli methionyl-tRNA synthetase. The structures of the corresponding complexes were solved at resolutions below 1.9A and compared to those of the enzyme free or complexed with methionine. Residues Y15 and W253 play key roles in the strength of the binding of the amino acid and of its analogues. Indeed, full motions of these residues are required to recover the maximum in free energy of binding. Residue Y15 also controls the size of the hydrophobic pocket where the amino acid side-chain interacts. H301 appears to participate to the specific recognition of the sulphur atom of methionine. Complexes with methionyl adenylate analogues illustrate the shielding by MetRS of the region joining the methionine and adenosine moieties. Finally, the structure of MetRS complexed to a methionine analogue mimicking the tetrahedral carbon of the transition state in the aminoacylation reaction was solved. On the basis of this model, we propose that, in response to the binding of the 3'-end of tRNA, Y15 moves again in order to deshield the anhydride bond in the natural adenylate.