Search

The preparation and structure–activity relationships (SARs) of potent and selective small molecule inhibitors of bacterial methionyl-tRNA synthetase (MetRS) derived from an oxazolone–dipeptide scaffold are described. Examples combine Staphylococcus aureus MetRS (SaMetRS) potency with selectivity over human MetRS. As a result of the SAR expansion compound 14a was identified, as a potent SaMetRS inhibitor (IC50=18 nM) having moderate inhibition of MetRS derived from Enterococci faecalis (IC50=3.51 μM).

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K(M) and k(cat) for aminoacylation of E. coli initiator tRNA(f)(Met) are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated.

Biotin is an essential cofactor of cell metabolism serving as a protein-bound coenzyme in ATP-dependent carboxylation, in transcarboxylation, and certain decarboxylation reactions. The involvement of biotinylated proteins in other cellular functions has been suggested occasionally, but available data on this are limited. In the present study, a Saccharomyces cerevisiae protein was identified that reacts with streptavidin on Western blots and is not identical to one of the known biotinylated yeast proteins. After affinity purification on monomeric avidin, the biotinylated protein was identified as Arc1p. Using 14C-labeled biotin, the cofactor was shown to be incorporated into Arc1p by covalent and alkali-stable linkage. Similar to the known carboxylases, Arc1p biotinylation is mediated by the yeast biotin:protein ligase, Bpl1p. Mutational studies revealed that biotinylation occurs at lysine 86 within the N-terminal domain of Arc1p. In contrast to the known carboxylases, however, in vitro biotinylation of Arc1p is incomplete and increases with BPL1 overexpression. In accordance to this fact, Arc1p lacks the canonical consensus sequence of known biotin binding domains, and the bacterial biotin:protein ligase, BirA, is unable to use Arc1p as a substrate. Arc1p was shown previously to organize the association of MetRS and GluRS tRNA synthetases with their cognate tRNAs thereby increasing the substrate affinity and catalytic efficiency of these enzymes. Remarkably, not only biotinylated but also the biotin-free Arc1p obtained by replacement of lysine 86 with arginine were capable of restoring Arc1p function in both arc1Delta and arc1Deltalos1Delta mutants, indicating that biotinylation of Arc1p is not essential for activity.

International audience; Yeast Arc1p, human p43 and plant methionyl-tRNA synthetase (MetRS) possess an EMAPII-like domain capable of non-specific interactions with tRNA. Arc1p interacts with MetRS (MES1) and GluRS and operates as a tRNA-interacting factor (tIF) in trans of these two synthetases. In plant MetRS, the EMAPII-like domain is fused to the catalytic core of the synthetase and acts as a cis-acting tIF for aminoacylation. We observed that the catalytic core of plant MetRS expressed from a centromeric plasmid cannot complement a yeast arc1 2 mes1 2 strain. Overexpression of the mutant enzyme from a high-copy number plasmid restored cell growth, suggesting that deletion of its C-terminal tIF domain was responsible for the poor aminoacylation efficiency of that enzyme in vivo. Accordingly, expression of full-size plant MetRS from a centromeric plasmid, but also of fusion proteins between its catalytic core and the EMAPII-like domains of yeast Arc1p or of human p43 restored cell viability. These data showed that homologous tIF domains from different origins are interchangeable and may act indifferently in trans or in cis of the catalytic domain of a synthetase. Unexpectedly, co-expression of Arc1p with the catalytic core of plant MetRS restored cell viability as well, even though Arc1p did not associate with plant MetRS. Because Arc1p also interacts with yeast GluRS, restoration of cell growth could be due at least in part to its role of cofactor for that enzyme. However, co-expression of human p43, a tIF that did not associate with plant MetRS or with yeast GluRS and MetRS, also restored cell viability of a yeast strain that expressed the catalytic core of plant MetRS. These results show that p43 and Arc1p are able to facilitate tRNA aminoacylation in vivo even if they do not interact physically with the synthetases. We propose that p43/Arc1p may be involved in sequestering tRNAs in the cytoplasm of eukaryotic cells, thereby increasing their availability for protein synthesis.

International audience; In class 1 aminoacyl-tRNA synthetases, methionyl-tRNA synthetases (MetRS) are homodimers or monomers depending on the presence or absence of a domain appended at the C-side of the polypeptide chain. Beyond this C-domain, all MetRS display a highly conserved catalytic core with a Rossmann fold, the two halves of which are linked by a connective peptide (CP). Three-dimensional folding of CP and its putative zinc content have served as a basis to propose a division of the MetRS family into four subgroups. All subgroups but one, which is predicted to display two zincs per MetRS polypeptide, have been characterized. In the present study, the 3D structure of MetRS from Pyrococcus abyssi could be solved at 2.9 A resolution. The data obtained and atomic absorption spectroscopic measurements establish the presence of two metal ions per polypeptide chain. This finding brings strong support to the above classification. In the crystal, the C-terminal dimerization domain is disordered. This observation is thought to reflect marked flexibility of the two core moieties with respect to the C-domains in the dimer. Gel shift experiments were performed with the isolated C-terminal dimerization domain and a core monomeric MetRS, both derived from the P. abyssi enzyme. Complex formation between the C-domain and the core enzyme could not be evidenced. Moreover, association of tRNA(Met) to the core enzyme is enhanced in the presence of the C-domain. Together, these experiments suggest positive control in trans by the C-domain on recognition of tRNA by the core moiety of MetRS.

The translation of mRNA during protein synthesis is among the most complex and conserved processes in the cell. The first step in translation is the attachment of the correct amino acid to its cognate tRNA, which is catalyzed by the ubiquitous aminoacyl-tRNA synthetase (aaRS) family of housekeeping enzymes. Although the catalytic activities of aaRSs are common among different species, there are distinct features that distinguish the higher eukaryotic versions from most of their prokaryotic counterparts. AaRSs are modular enzymes composed of conserved catalytic cores and additional appended domains acquired during evolution.1,2 These domains are required for RNA recognition and proofreading activities and are also involved in protein–protein interactions and various other roles, thereby expanding aaRS functions beyond translation of mRNA. One of the most important roles of appended domains in higher eukaryotic and some archaeal aaRSs is to mediate the formation of intricate networks of protein–protein interactions between different components of the translation machinery and other components of the cell.3 AaRSs have been shown to form a variety of complexes with each other and other components of the cell, one of the most complex and intriguing of which is the mammalian multi-synthetase complex (MSC). Among the 20 aaRSs, the mammalian MSC is composed of 9 aaRSs (ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, and ProRS) that associate with three auxiliary protein factors AIMP1 (p43), AIMP2 (p38), and AIMP3 (p18) [aaRS-interacting multifunctional protein (AIMP)].4 Smaller aaRS‐containing complexes have also been observed in lower eukaryotes and archaea.3 Within the mammalian MSC, AIMP1 increases the catalytic activity of ArgRS, AIMP2 interacts with most of the MSC components and is considered an essential scaffolding protein in the complex, but the role of AIMP3 remained unknown.5 A recent study of Caenorhabditis elegans showed that different MSC structures result from both additions and subtractions to aaRS cores and changes in AIMP content during evolution. It was observed that two clades of the bilaterian phylum reflect divergent evolution of the MSC, with the notable difference in arthropods being the absence of a fused Glu-ProRS, presence of ValRS instead of AspRS, presence of a p43 (AIMP1)-like domain fused to the C-terminus of MetRS, and absence of AIMP3.6 These last two findings are of particular interest as they emphasize the importance of retaining MetRS in the MSC, which, in higher eukaryotes, occurs via direct interactions with AIMP3.7 AIMP3 has significant sequence similarity to eukaryotic elongation factors, which raises questions as to how interactions between AIMP3 and MetRS in the MSC might impact translation.8 In this issue, Kang et al. now show that AIMP3, rather than just being a scaffolding protein, plays a central role in translation by mediating the transfer of aminoacylated initiator tRNA from MetRS to the elongation initiation factor 2 (eIF2) complex. Kang et al. reasoned that as AIMP3 both interacts strongly with MetRS in the MSC and has sequence similarity to elongation factors, it was likely to have a direct role in translation. Consistent with this prediction, they found that AIMP3 directly bound a form of tRNA, Met-tRNAiMet, that is reserved exclusively for translation initiation. A possible role in translation initiation was further supported by the fact that the corresponding tRNA used for translation elongation, Met-tRNAeMet, did not bind AIMP3. The specificity displayed by AIMP3 for methionine-charged initiator tRNAs is also a hallmark of eIF2γ, a subunit of eIF2 involved in tRNA binding during translation initiation complex formation.9,10 Kang et al. then went on to show that AIMP3 recruits eIF2γ to MetRS, consistent with the overlapping tRNA binding activities of the three proteins. These interactions require both Met-tRNAiMet and the active form of eIF2γ, the GTP binding domain of which interacts with MetRS and AIMP3. These findings together provide a model for the role of the MSC in initiation complex formation, the G domain of eIF2γ interacts with AIMP3-Met-tRNAiMet-MetRS, followed by transfer of Met-tRNAiMet to the groove formed between the G domain and domain II of eIF2γ. After the transfer of Met-tRNAiMet, domain III of eIF2γ can then interact with helix 44 of the 40S ribosome leading to formation of the 43S pre-initiation complex.9 The authors provided preliminary support for this model using an AIMP3 knockdown, which caused a 40% reduction in global translation and changes to the co-localization of eIF2γ with ribosomes. While further studies are now required to further confirm the precise details of AIMP3 function, it is clear from the study of Kang et al. that it plays a pivotal role in maintaining the efficiency of translation initiation in higher eukaryotes. The study by Kang et al. not only advances our understanding of AIMP3 function but also provides perhaps the most direct evidence to date that the higher eukaryotic MSC has a critical role in optimizing translation in addition to its known regulatory functions.11,12 MSC formation has been implicated in a number of roles as a depot for releasable regulatory proteins, in which the aaRSs and auxiliary factors maintain ordinary activity but acquire new functions upon release from the complex.11,13 The findings of Kang et al. and other recent studies from a number of laboratories are now starting to show that the roles of MSC components and aaRSs inside and outside translation may actually be quite intricately intertwined.14,15 For example, under UV stress, phosphorylation by GCN2 reduces the activity of MetRS, thereby reducing translation initiation16 and providing an additional level of control of AIMP3-mediated translation initiation. This, in turn, raises questions of how and under what circumstances AIMP3 itself is regulated. Whatever the answer is, the work of Kang et al. described in this issue adds significantly to our understanding of how aminoacyl-tRNA synthesis is linked to an increasing number of key cellular processes both inside translation and outside translation.

The genetic code is based on specific interactions between transfer RNA (tRNA) synthetases and their cognate tRNAs. The anticodons for methionine and isoleucine tRNAs differ by a single nucleotide, and changing this nucleotide in an isoleucine tRNA is sufficient to change aminoacylation specificity to methionine. Results of combinatorial mutagenesis of an anticodon-binding-helix loop peptide were used to design a hybrid sequence composed of amino acid residues from methionyl- and isoleucyl-tRNA synthetases. When the hybrid sequence was transplanted into isoleucyl-tRNA synthetase, active enzyme was generated in vivo and in vitro. The transplanted peptide did not confer function to methionyl-tRNA synthetase, but the substitution of a single amino acid within the transplanted peptide conferred methionylation and prevented isoleucylation. Thus, the swap of a single amino acid in the transplanted peptide switches specificity between anticodons that differ by one nucleotide.

Starting with a micromolar lead identified from high-throughput screening, a series of pyrazoles were discovered with significantly improved potency on bacterial methionyl-tRNA synthetase and selectivity over human methionyl-tRNA synthetase.

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.

The screening of new antibiotics against several bacterial strains often reveals unexpected occurrences of natural drug resistance. Two examples of this involve specific inhibitors of Staphylococcus aureus isoleucyl-transfer-RNA synthetase 1 (IleRS1) and, more recently, Streptococcus pneumoniae methionyl-tRNA synthetase 1 (MetRS1). In both cases, resistance is due to the presence of a second gene that encodes another synthetase (IleRS2 or MetRS2). Here, we show that both S. pneumoniae MetRS2 and S. aureus IleRS2 have closely related homologues in the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Furthermore, similar to drug-resistant pathogens, strains of B. anthracis and its closest relative, B. cereus, also have wild-type ileS1 and metS1 genes. Clostridium perfringens, the causative agent of gangrene, also has two metS genes, whereas Oceanobacillus iheyensis isolated from deep-sea sediments has a single ileS2-type gene. This study shows the importance of understanding complex evolutionary networks of ancient horizontal gene transfer for the development of novel antibiotics.

Conformationally restricted analogues of the central linker unit of bacterial methionyl tRNA synthetase (MRS) inhibitors have been prepared. The (1S,2R)-cyclopentylmethyl moiety was identified as the preferred cyclic linker, with significant diastereo- and enantioselectivity of activity. Combination of this linker with an optimal substituted aryl right-hand side has resulted in a compound with exceptionally good antibacterial activity against staphylococci and enterococci, including antibiotic resistant strains.

International audience; The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.

FREE FULL TEXT http://www.proteinscience.org/cgi/content/full/11/12/2871; Protein Blocks (PBs) comprise a structural alphabet of 16 protein fragments, each 5 Calpha long. They make it possible to approximate and correctly predict local protein three-dimensional (3D) structures. We have selected the 72 most frequent sequences of five PBs, which we call Structural Words (SWs). Analysis of four different protein data banks shows that SWs cover 92% of the amino acids in them and provide a good structural approximation for residues (i.e., sequences) 9 Calpha long. We present most of them in a simple network that describes 90% of the overall residues and, interestingly, includes more than 80% of the amino acids present in coils. Analysis of the network shows the specificity and quality of the 3D descriptions as well as a new type of relation between local folds and amino acid distribution. The results show that the 3D structure of these protein data banks can be easily described by a combination of subgraphs included in the network. Finally, a Bayesian probabilistic approach improved the prediction rate by 4%.

The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

Methionyl-tRNA synthetase (MetRS) belongs to the family of 20 enzymes essential for protein biosynthesis. It links covalently methionine with its cognate tRNA. Crystal structures solved for bacterial MetRSs have given a number of interesting insights into enzyme architecture and methionylation catalysis. A comparison of sequences of MetRSs belonging to all kingdoms of life, as well as numerous biochemical and genetic studies have revealed the presence of various additional domains appended to the catalytic core of synthetase. They are responsible for interactions with tRNA and proteins. Tertiary structure of C-terminal tRNA-binding appendices can be deduced from those determined for their homologues: tRNA binding protein 111 and endothelial monocyte-activating polypeptide II. Contacts between MetRS and other proteins could be mediated not only by noncatalytic peptides but also by structural elements present in the catalytic core, e.g. Arg-Gly-Asp (RGD) motifs. Additional activities involve MetRS in the maintenance of translational fidelity and in coordination of ribosome biogenesis with protein synthesis.

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.

The structure activity relationship on a series of ester and hydroxamate analogues of methionyl and isoleucyl adenylate has been investigated through introducing linkers between the 1'-position of ribose and adenine surrogates as methionyl-tRNA, and isoleucyl-tRNA synthetase inhibitors, respectively. The results indicate that ester analogue 23 was found to be a potent inhibitor of Escherichia coli methionyl-tRNA synthetase, and its interaction with the active site was proposed by a molecular modeling study.

Amino acid selection by aminoacyl-tRNA synthetases requires efficient mechanisms to avoid incorrect charging of the cognate tRNAs. A proofreading mechanism prevents Escherichia coli methionyl-tRNA synthetase (EcMet-RS) from activating in vivo l -homocysteine, a natural competitor of l -methionine recognised by the enzyme. The crystal structure of the complex between EcMet-RS and l -methionine solved at 1.8 A resolution exhibits some conspicuous differences with the recently published free enzyme structure. Thus, the methionine δ-sulphur atom replaces a water molecule H-bonded to Leu13N and Tyr260Oη in the free enzyme. Rearrangements of aromatic residues enable the protein to form a hydrophobic pocket around the ligand side-chain. The subsequent formation of an extended water molecule network contributes to relative displacements, up to 3 A, of several domains of the protein. The structure of this complex supports a plausible mechanism for the selection of l -methionine versus l -homocysteine and suggests the possibility of information transfer between the different functional domains of the enzyme.

International audience; 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 epsilon-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.

Background: The 20 aminoacyl-tRNA synthetases are divided into two classes, I and II. The 10 class I synthetases are considered to have in common the catalytic domain structure based on the Rossmann fold, which is totally different from the class II catalytic domain structure. The class I synthetases are further divided into three subclasses, a, b and c, according to sequence homology. No conserved structural features for tRNA recognition by class I synthetases have been established. Results: We determined the crystal structure of the class Ia methionyl-tRNA synthetase (MetRS) at 2.0 A resolution, using MetRS from an extreme thermophile, Thermus thermophilus HB8. The T. thermophilus MetRS structure is in full agreement with the biochemical and genetic data from Escherichia coli MetRS. The conserved ‘anticodon-binding' residues are spatially clustered on an α-helix-bundle domain. The Rossmann-fold and anticodon-binding domains are connected by a β–α–α–β–α topology (‘SC fold') domain that contains the class I specific KMSKS motif. Conclusions: The α-helix-bundle domain identified in the MetRS structure is the signature of the class Ia enzymes, as it was also identified in the class Ia structures of the isoleucyl- and arginyl-tRNA synthetases. The β–α–α–β–α topology domain, which can now be identified in all known structures of the class Ia and Ib synthetases, is likely to dock with the inner side of the L-shaped tRNA, thereby positioning the anticodon stem.

Engineered aminoacyl-tRNA synthetases have been used to enable the incorporation of many unnatural amino acids into recombinant proteins in vivo. In the majority of these studies, the engineered synthetase is harbored on a plasmid while the host retains a wild-type copy of the synthetase in its genome. Herein, we construct a strain carrying a single genomic copy of a methionyl-tRNA synthetase (MetRS) gene, metG*, engineered to enable the incorporation of azidonorleucine (ANL) into proteins. The resulting strain, M15MA metG*, is capable of both supporting robust cell growth and enabling the production of20 mg/L culture of a recombinant protein, murine dihydrofolate reductase, containing ANL. The extent of replacement of methionine with ANL in this protein is 90%. Using this strain, we also produce ANL-containing OmpC, an outer membrane protein, and demonstrate that the surface of cells displaying this protein can be covalently modified using copper-catalyzed azide-alkyne cycloaddition. Since this mutant MetRS has been introduced into the genome, as opposed to a plasmid, M15MA metG* is genetically stable.

International audience; The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.

We report here evidence for mutations that break domain-domain functional communication in a synthetase-tRNA complex. Each synthetase is roughly divided into two major domains that are paralleled by the two arms of the L-shaped tRNA structure. The active-site-containing domain interacts with the acceptor arm of the tRNA. The second domain frequently interacts with the anticodon-containing arm. By an induced-fit mechanism, contacts with the anticodon can activate formation of a robust transition state at a site over 70 A away. This induced-fit-based activation is thought to occur through domain-domain signaling and is seen by the enhancement of aminoacylation of the anticodon-containing full tRNA versus a substrate based on the acceptor arm alone. Here we describe a rationally designed mutant methionyl-tRNA synthetase containing two point substitutions at sites that potentially link an anticodon-binding motif to the catalytic domain. The double mutation had no effect on interactions with either the isolated acceptor arm or the anticodon stem-loop. In contrast to interactions with the separate pieces, the mutant enzyme was severely impaired for binding the native tRNA and lost much of its ability to enhance the rate of charging of the full tRNA over that of a substrate based on the acceptor arm alone. We propose that these residues are part of a network for facilitating domain-domain communication for formation of an active synthetase-tRNA complex by induced fit.

The transcription factor CHOP is involved in the regulation of the cell division cycle and the control of programmed cell death in response to cellular stress. CHOP expression has been linked with several forms of cancer. A reciprocal translocation between the CHOP and TLS RNA-binding protein gene results in myxoid liposarcoma and amplifications of the CHOP gene are associated with solid tumors including several types of sarcomas. Here we report the mapping of the methionyl tRNA synthetase (MetRS) gene to the identical 12q13 locus where the CHOP gene had previously been mapped. PCR analysis demonstrates a tail-to-tail overlap of both genes over a 55-bp region. As a result the two mRNAs share a 3' UTR complementary sequence allowing an in vivo interaction between the two mRNAs. An AU-rich regulatory element (ARE) known to control mRNA stability resides in the overlapping sequence. To test for functional significance of the ARE a luciferase reporter plasmid containing the 3'UTR of CHOP was constructed. Transfection experiments in NIH-3T3 cells show that CHOP 3'UTR confers a significantly lower activity than a control reporter or a reporter in which the region overlapping the MetRS mRNA is deleted. The conservation of this overlapping of the CHOP and MetRS genes and the role of their complementary sequence in the control of mRNA stability suggest the existence of a functional link between the expression of these two genes.

Interactions of specific amino acid residues of the carboxyl-terminal domain of MetRS with the CAU anticodon of tRNAMet assure accurate and efficient aminoacylation. The substitution of one such residue, Trp461 by Phe, impairs the binding of cognate tRNA, but enhances the binding of noncognate tRNAs, particularly those containing G at the wobble position. However, the enhanced binding of noncognate tRNAs is not accompanied by the increased aminoacylation of these tRNAs. A genetic screening procedure was designed to isolate methionyl-tRNA synthetase mutants which were able to aminoacylate a GGU (threonine) anticodon derivative of tRNAfMet. One such mutant, obtained from W461F MetRS, had an Ile29 to Thr substitution in helix A located in the amino-terminal dinucleotide-fold domain that forms the site for amino acid activation. Analysis of the catalytic properties of the I29T/W461F enzyme indicates that the mutation in helix A of the dinucleotide-fold domain affects kcat for aminoacylation of tRNAs having a GGU threonine anticodon. Interactions with cognate tRNAfMet (CAU), as well as with methionine and ATP were not affected by the Ile29 to Thr substitution. We conclude that the I29T substitution leads to a slight adjustment of the alignment of the CCA stem of noncognate tRNAs (GGU) in the catalytic domain of the enzyme, reflected in the increase in kcat, which also allows mischarging in vivo. A function of Ile29 is therefore to minimize the mischarging of tRNAThr (GGU) by methionyl-tRNA synthetase. The methods described here provide useful tools for examining the mechanisms of tRNA selection by aminoacyl-tRNA synthetases.

A genomic sequence encoding mitochondrial methionyl-tRNA synthetase (MetRS) was determined from a pathogenic fungi Candida albicans. The gene is distinct from that encoding the cytoplasmic MetRS. The encoded protein consists of 577 amino acids (aa) and contains the class I defining sequences in the N-terminal domain and the conserved anticodon-binding amino acid, Trp, in the C-terminal domain. This protein showed the highest similarity with the mitochondrial MetRSs of Saccharomyces cerevisiae and Shizosaccharomyces pombe. The mitochondrial MetRSs of these fungi were distinguished from their cytoplasmic forms. The protein lacks the zinc binding motif in the N-terminal domain and the C-terminal dimerization appendix that are present in MetRSs of several other species. Escherichia coli tRNAMet was a substrate for the encoded protein as determined by genetic complementation and in vitro aminoacylation reaction. This cross-species aminoacylation activity suggests the conservation of interaction mode between tRNAMet and MetRS.

Methionine tRNA was purified from muscle mitochondria of the ascidian Halocynthia roretzi and its RNA sequence was determined. Analysis of the nucleotide sequence revealed that unlike most metazoan mitochondrial tRNAs(Met), which have a highly conserved cytidine (C) or C-derivative at the wobble position, the H. roretzi mitochondrial tRNA(Met) possesses 5-carboxymethylaminomethyluridine (cmnm5U) at the first position of the anticodon. This is the first report of a single mitochondrial tRNA(Met) species having uridine (U) or a U-derivative at the wobble position.

The effect of abnormal proteins on cell viability was studied using artificially cleaved polypeptides.Escherichia colimethionyl-tRNA synthetase (MetRS) consists of two distinct domains and its activity is essential for cell viability. The polypeptide chain was split by linker insertion and expressed as two fragments. Two pairs of polypeptides, one split within the N-terminal domain and another at the junction of the two domains retained aminoacylation activity. Thein vitroactivities of these split mutants were enhanced by the presence of chaperonin, GroESL. However, cells containing these split polypeptides became sensitive to conditions that induce GroESL. The results of this work suggest that an abnormally generated protein can cause cell death under stressful conditions.

Activation of amino acid homocysteine was compared with that of methionine in rabbit crude liver extracts and purified multi-enzyme complex of aminoacyl-tRNA synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacetic-acid insoluble materials in the absence of protein synthesis. Homocysteine synthetase activity was found in the crude extract and in the purified multi-enzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five times higher than the activation of homocysteine. There was a partial loss of Hcy-tRNA synthetase activity in the purified multi-enzyme complex. Preliminary reconstitution experiments indicated a requirement for an additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [14C]homocysteine, revealed radioactivity in homocysteine, methionine and homocysteine thiolactone, indicating a conversion of tRNA-attached homocysteine to methionine. Total tRNA was separated on a benzoylated cellulose column into a fraction enriched in initiator tRNA and a methionine-accepting, but initiator tRNA-deficient, fraction. Homocysteine-accepting activity was present only in the initiator tRNA-enriched fraction. Based on the above data we propose that homocysteine activation in reticulocyte lysates, reported previously, also occurs in liver. Activated homocysteine is attached to initiator tRNA and then converted to methionine by a methylating enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.

Escherichia coli methionyl-tRNA synthetase consists of catalytic, anticodon-binding, and dimerization domains. The polypeptide was genetically cleaved and expressed as multiple subunits to investigate how peptide breakage affects the activity and stability of the enzyme. Mutants cleaved near conserved or functionally important sites were inactive. A few bipartite mutants were active, but they showed temperature sensitivity in their activity and stability. An additional cleavage of the active bipartites inactivated the enzyme, suggesting that at least two functional domains have to be covalently connected to retain the activity. This approach proves to be useful in determining the structural and functional organization of a protein.

International audience; Alignment of the sequences of methionyl-tRNA synthetases from various microbial sources shows low levels of identities. However, sequence identities are clustered in a limited number of sites, most of which contain peptide patterns known to support the activity of the Escherichia coli enzyme. In the present study, site-directed mutagenesis was used to probe the role of these conserved residues in the case of the Bacillus stearothermophilus methionyl-tRNA synthetase. The B. stearothermophilus enzyme was chosen in this study because it can be produced as an active truncated monomeric form, similar to the monomeric derivative of E. coli methionyl-tRNA synthetase produced by mild proteolysis. The two core enzyme molecules share only 27% identical residues. The results allowed the identification of the binding sites for ATP, methionine and tRNA, as well as that responsible for the tight binding of the zinc ion to the enzyme. It is concluded that the thermostable synthetase adopts a three-dimensional folding very similar to that of the E. coli one. Therefore, the two methionyl-tRNA synthetase sequences, although significantly different, maintain a common scaffold with the functionally important residues exposed at constant positions. Sequence alignments suggest that the above conclusion can be generalized to the known methionyl-tRNA synthetases from various sources.

Sequence analysis of an 11,628 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII revealed the presence of the 5' end of the RAD2 gene, the MES1 gene and six open reading frames (ORFs) each longer than 300 bp. Four of these ORFs are expressed genes, as indicated by transcript analysis. One of them, YGR261c, which specifies a putative beta-adaptine, corresponds to gene YKS5, which has recently been identified as a suppressor of loss of casein kinase 1 function. The remaining three ORFs are new genes; of these, YGR260w encodes a protein showing similarity to the S. cerevisiae allantoate permease and YGR262c specifies a putative protein kinase.

To obtain direct evidence for the attachment of 5SrRNA-ribosomal L5 protein particles (5SRNP) and methionine-tRNA (tRNA(met)) to methionyl-tRNA synthetase (MetRS) in the macromolecular aminoacyl-tRNA synthetase (ARS) complex of rat liver, a MetRS-5SRNP-tRNA(met) complex was dissociated from the macromolecular ARS complex fraction by n-octyl-beta-D-glucoside (Method I) or by omega-aminooctyl agarose (Method II) chromatography. The dissociated MetRS complex fraction was purified by gel filtration followed by tRNA-Sepharose chromatography using partially purified tRNA(met) in Method I, and by hydrophobic interaction chromatography in Method II. In both methods, final Superdex200 chromatography showed that MetRS activity was present in the region corresponding to the molecular weight of the MetRS-5SRNP-tRNA(met) complex (M(r) 200,000). One main protein band corresponding to the molecular weight of MetRS was observed on SDS-PAGE of the final product, which was concentrated by lyophilizing after dialysis against water. Using serum albumin as an inhibitor of adhesion of L5 to the microconcentrators which was used to concentrate the final product, a distinct L5 band was detected on SDS-PAGE, the intensity of which was comparable to that of the MetRS band. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 5SrRNA. Dot blot analysis using an antibody against ribosomal protein L5 showed that L5 was present in the Superdex200 fractions prepared by both methods. The MetRS specific activities in MetRS complex fractions incubated without tRNA increased during the purification procedures, indicating that endogeneous tRNA(met) exists stably in the MetRS complex. 5SRNP and 5SrRNA markedly enhanced the MetRS activity in the MetRS complex, indicating that 5SRNP(A) plays a role as a positive effector of MetRS.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

The class I methionyl tRNA synthetase has a conserved N-terminal nucleotide binding fold which contains the active site, and a largely non-conserved C-terminal anticodon binding domain. At the C-terminal end of the anticodon binding domain is a peptide which curls back into the N-terminal nucleotide binding fold near the active site. We showed that a mutation in this peptide disrupts aminoacylation and binding of a 7 base pair microhelix substrate based on the acceptor stem of tRNAfMet. The novel technique of affinity coelectrophoresis was applied to this system for the first time to determine dissociation constants of wild-type and mutant MetRS for small RNA substrates. A description and evaluation of this technique for measuring weak protein-nucleic acid interactions is presented here, in the context of the methionyl tRNA synthetase system.

Determinants for the identities of tRNAs are located in the acceptor stem and, commonly, in the anticodon as well. Although the anticodon is an important determinant for the identity of methionine tRNA, RNA microhelices whose sequences are based on the acceptor stem alone can be aminoacylated by the class I methionyl-tRNA synthetase. We show here that specific nucleotide substitutions in a microhelix significantly reduced its rate of aminoacylation. In contrast, affinity coelectrophoresis analysis showed that microhelix binding to the enzyme was not significantly affected by the same substitutions. These and additional experiments and considerations imply that specific determinants for microhelix aminoacylation are needed for orientation of the acceptor stem in the transition state of catalysis rather than for enhanced binding interactions. The effect of linking together acceptor stem interactions with those in the anticodon, as occurs in the whole tRNA molecule, was also evaluated. This analysis showed that linkage results in some of the favorable acceptor stem and anticodon interactions being used to offset the free energy cost of straining the structure of the enzyme-tRNA complex.

Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components. These macromolecules interact in a specific and concerted manner to yield the translation initiation complex. This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process. The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA f Met formyltransferase are described. In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered. Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process.

Rat liver cytosol was incubated with a trace amount of rat liver 5SrRNA which was highly labeled at the 3'-end with cytidine 3',5'-[5'-32P]biphosphate, and with [35S]methionine in the presence of ATP mixture, and then with an antibody against ribosomal protein L5. The mixture was analyzed by protein A-Sepharose chromatography. The following results were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) from the protein A-Sepharose column contained an overlapping peak of 32P- and 35S-radioactivities. In a control experiment using the same amount of 32P-labeled Escherichia coli 5SrRNA with the same specific activity, no fraction of the eluate contained 32P-radioactivity. (ii) The fractions containing both 32P- and 35S-radioactivities from the protein A-Sepharose column were crosslinked by UV irradiation. The products was subjected to PAGE, and RNA in each gel slice was eluted and purified. The fraction containing both 32P- and 35S-radioactivities was present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low 32P- and 35S-radioactivities were present in this region in the control experiment without UV irradiation. This finding suggested that [35S]methionyl-tRNA interacted with 32P-labeled 5SRNP. (iii) The fraction containing overlapping 32P- and 35S-radioactivities described above was subjected to Sephadex G-150 chromatography. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10,000 to 250,000 with a peak at about 200,000, suggesting the presence of a complex containing Met-RS (Mr 108,000), 5SRNP (Mr 74,000), and methionyl-tRNA (Mr 25,000). Furthermore, this fraction showed definite Met-RS activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS). The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the alpha-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate. The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS.

In many cases, the intrinsic binding energies of amino acids to aminoacyl-tRNA synthetases are inadequate to give the required accuracy of translation. This has necessitated the evolution of a second determinant of specificity, proofreading, or editing mechanisms that involve the expenditure of energy to remove errors. Studies of an error-editing function of bacterial methionyl-tRNA synthetase have led to the discovery of a distinct chemical mechanism of editing and to molecular dissection of the dual synthetic-editing function of the active site of the synthetase. Studies have also established the importance of proofreading in living cells and allowed direct measurements of energy costs associated with editing in vivo. An unexpected outcome of these studies was a discovery of functional and structural similarities between methionyl-tRNA synthetase and S-adenosylmethionine synthetase, suggesting an evolutionary relationship between the two proteins. The mechanism of editing involves a nucleophilic attack of a sulfur atom on the side chain of homocysteine in homocysteinyl adenylate on its carbonyl carbon, yielding homocysteine thiolactone. The model of the active site of methionyl-tRNA synthetase derived from structure-function studies explains how the active site partitions amino acids between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions of active site residues Trp305 and Tyr15 with the side chain of methionine prevent the cognate amino acid from entering the editing pathway. These interactions are missing in the case of the smaller side chain of the noncognate homocysteine, which therefore enters the editing pathway. Homocysteine thiolactone is formed as a result of editing of homocysteine by methionyl-tRNA synthetase in bacteria, yeast, and some cultured mammalian cells. In mammalian cells, enhanced synthesis of homocysteine thiolactone, is, thus far, associated with oncogenic transformation. In E. coli, most of the energy cost of proofreading by methionyl-tRNA synthetase is due to editing of the incorrect product, homocysteinyl adenylate.

The synthetic and editing functions of three forms of Escherichia coli methionyl-tRNA synthetase with different C-terminal sequences have been compared in vivo and in vitro. These forms include a full-length wild-type dimer (MRS676), a truncated monomer (MRS547) believed to be equivalent to the biologically active large tryptic fragment, and a third form denoted MRS581*. DNA sequencing revealed that MRS581* is predicted to contain 18 additional amino acids from the wild-type full-length sequence at the carboxy terminus of truncated form MRS547, and this is then fused to an additional 16 amino acids encoded by vector pBR322. Both MRS676 and MRS581* were found to edit endogenous homocysteine about 20-fold more efficiently than MRS547 in vivo. However, the three methionyl-tRNA synthetases edited exogenously supplied homocysteine in bacterial cultures to similar extents. Purified proteins exhibited no significant differences in editing function in vitro. Synthetic activity of purified MRS676 in vitro was found to be about 2.5-fold higher per subunit compared to the shorter forms of the enzyme. The C-terminal region in E. coli methionyl-tRNA synthetase is thus suggested to play an important role in editing in vivo, most likely by allowing interaction of the enzyme with the methionine biosynthetic pathway. These data support a model of channeling of at least some metabolites in bacterial protein synthesis.

A program is described for estimating enzymatic parameters from experimental data using Apple Macintosh computers. MC-Fit uses iterative least-square fitting and Monte-Carlo sampling to get accurate estimates of the confidence limits. This approach is more robust than the conventional covariance matrix estimation, especially in cases where experimental data is partially lacking or when the standard error on individual measurements is large. This happens quite often when analysing the properties of variant enzymes obtained by mutagenesis, as these can have severely impaired activities and reduced affinities for their substrates.

The isotopic [32P]PPi-ATP exchange activity of isoleucyl-, valyl-, histodyl-, tyrosyl-, and methionyl-tRNA synthetases from Escherichia coli are lost upon incubation in the presence of pyridoxal-5′-phosphate (PLP). When the residual activity of either isoleucyl-, valyl-, or methionyl-tRNA synthetase (monometric truncated form) was plotted as a function of the number of PLP molecules incorporated per enzyme molecule, the plots obtained appeared biphasic. Below 50% inactivation of these enzymes, PLP incorporation varied linearly with the isotopic exchange measurements, and extrapolation of the first half of the plot indicated a stoichiometry of 1.10±0.05 mol of PLP incorporated per mol of 100% inactivated synthetase. Beyond 50% inactivation, the graph deviated from its initial slope, and up to 4–5 mol of PLP were incorporated per mol of synthetase at the highest used PLP concentrations. In the cases of homodimeric histidyl- and tyrosyl-tRNA synthetases, extrapolation of the graph at 100% inactivation indicated 2.8 ± 0.1 and 2.4±0.1 mol of PLP incorporated per mol of enzyme, respectively, PLP-labeled peptides were obtained through trypsin digestion and RPLC purification, prior to Edman degradation analysis. PLP-labeled residues were identified as lysines 132, 332, 335 and 402 of monomeric methionyl-tRNA synthetase, lysines 332, 335, 402, 465, 596 and 640 of native dimeric methionyl-tRNA synthetase, lysines 22, 117, 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559, 593 and 909 of valyl-tRNA synthetase, lysines 2, 118, 369 and 370 of histidyl-tRNA synthetase and lysine 237 of tyrosyl-tRNA synthetase. In addition, the amino terminal residue of the polypeptide chain(s) of either isoleucyl-, valyl-, histidyl- or methionyl-tRNA synthetases was found labeled. Among these residues, lysines 332, 335 and 402 of monomeric methionyl-tRNA synthetase as well as lysines 332, 335, 402 and 596 of dimeric methionyl-tRNA synthetase, lysines 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559 of valyl-tRNA synthetase, lysines 2, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase were labeled in the presence of PLP concentrations smaller than or equal to 1 mM, and are shown to be critical for the activity of the enzymes. It is concluded that these residues participate to the binding sites of the phosphates of ATP on the studied synthetases.

Matrix assisted laser desorption mass spectrometry provides a very fast and efficient way to check protein sequences by the analysis of whole proteolytic digests. This method has been applied to the sequence verification of E. coli isoleucyl tRNA synthetase, for which two different sequences are found in data banks. The result of this investigation clearly shows that no one of these sequences is correct.

The 10 class I tRNA synthetases have an N-terminal nucleotide-binding fold which contains the catalytic center. Insertions into the nucleotide-binding fold provide contacts for acceptor-helix interactions, which stabilize the amino acid acceptor end of the tRNA substrate in the active site. A separate and largely nonconserved C-terminal domain provides contacts with distal parts of the tRNA, including the anticodon. For Escherichia coli methionyl tRNA synthetase, whose structure is known, the C-terminal domain is predominantly alpha-helical and forms a loop which interacts with the anticodon trinucleotide located about 76 A from the amino acid attachment site. Fused to the end of this helical domain is a peptide which curls back into the N-terminal nucleotide-binding fold and region of the active site. We show here that mutations in this peptide appendix disrupt aminoacylation and binding of a 7 base pair microhelix substrate based on the acceptor stem of tRNA(fMet), without affecting interactions with ATP or methionine or with the tRNA(fMet) anticodon. The impairment of acceptor-helix interactions by mutation of the C-terminal peptide can offset favorable anticodon interactions and severely reduce aminoacylation of tRNA(fMet). Thus, in addition to, or as an alternative to, acceptor-helix-binding insertions into the N-terminal nucleotide-binding fold, C-terminal peptide epitopes in some class I enzymes may provide a mechanism for facilitating RNA microhelix interactions with the catalytic site.