Michel Panvert

Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet.

The crystal structure of Escherichia coli methionyl‐tRNAfMet transformylase complexed with formyl‐methionyl‐tRNAfMet was solved at 2.8 Å resolution. The formylation reaction catalyzed by this enzyme irreversibly commits methionyl‐tRNAfMet to initiation of translation in eubacteria. In the three‐dimensional model, the methionyl‐tRNAfMet formyltransferase fills in the inside of the L‐shaped tRNA molecule on the D‐stem side. The anticodon stem and loop are away from the protein. An enzyme loop is wedged in the major groove of the acceptor helix. As a result, the C1‐A72 mismatch characteristic of the initiator tRNA is split and the 3′ arm bends inside the active centre. This recognition mechanism is markedly distinct from that of elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T‐stem side.

Structural Basis of RNA-Dependent Recruitment of Glutamine to the Genetic Code

Glutaminyl–transfer RNA (Gln-tRNAGln) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNAGln by the heterodimeric Glu-tRNAGln amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNAGln at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNAGln mutants characterized the catalytic centers for the enzymes three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom–long channel for ammonia transport connects the active sites in GatD and GatE. tRNAGln recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.

Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase-β-galactosidase chimeric protein

The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

Structure of the ternary initiation complex aIF2-GDPNP- methionylated initiator tRNA

Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that has a crucial role in the selection of the correct start codon on messenger RNA. We report the 5-Å resolution crystal structure of the ternary complex formed by archaeal aIF2 from Sulfolobus solfataricus, the GTP analog GDPNP and methionylated initiator tRNA. The 3D model is further supported by solution studies using small-angle X-ray scattering. The tRNA is bound by the α and γ subunits of aIF2. Contacts involve the elbow of the tRNA and the minor groove of the acceptor stem, but not the T-stem minor groove. We conclude that despite considerable structural homology between the core γ subunit of aIF2 and the elongation factor EF1A, these two G proteins of the translation apparatus use very different tRNA-binding strategies.

Structural bases for 16 S rRNA methylation catalyzed by ArmA and RmtB methyltransferases

Aminoglycosides are used extensively for the treatment of severe infections due to Gram-negative bacteria. However, certain species have become highly resistant after acquisition of genes for methyltransferases which catalyze post-transcriptional methylation of N7-G1405 in 16 S rRNA of 30 S ribosomal subunits. Inactivation of this enzymatic activity is therefore an important challenge for development of an effective therapy. The present work describes the crystallographic structures of methyltransferases RmtB and ArmA from clinical isolates. Together with biochemical experiments, the 3D structures indicate that the N-terminal domain specific for this family of methyltransferases is required for enzymatic activity. Site-directed mutagenesis has enabled important residues for catalysis and RNA binding to be identified. These high-resolution structures should underpin the design of potential inhibitors of these enzymes, which could be used to restore the activity of aminoglycosides against resistant pathogens.

Transcription and regulation of expression of the Escherichia coli methionyl-tRNA synthetase gene

SummaryThe DNA sequence and transcriptional organization around the Escherichia coli methionyl-tRNA synthetase gene, metG, were resolved. This gene can be transcribed in vivo and in vitro from two distinct promoters separated by 510 nucleotides. The upstream promoter is located within the coding sequence of a divergent gene expressing a protein of Mr 39 kDa of unknown function. Transcription originating from this upstream promoter is attenuated by a Rho-independent terminator before entering the structural gene. This leader RNA contains several potentially stable secondary structures, one of which shows striking similarity to tRNAMet, but no methionine-rich coding sequence. The regulation of metG expression was investigated by means of fusions to the lacZ gene. Transcription of a metG: : lacZ fusion is induced in a metG mutant and, reciprocally, repression is observed in a methionyl-tRNA synthetase overproducing strain. A model of metG expression control is proposed.

Recognition of tRNAs by Methionyl-tRNA Transformylase from Mammalian Mitochondria*

Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator. In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNAMet. In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA f Met mainly involves a set of nucleotides in the acceptor stem, particularly a C1A72 mismatch. In animal mitochondria, only one tRNAMet species has yet been described. It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group. In the present study, we searched for the identity elements of tRNAMet that govern its formylation by bovine mitochondrial transformylase. The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate. Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNAMet an A1U72motif.

Methionyl-tRNA synthetase from Bacillus stearothermophilus : structural and functional identities with the Escherichia coli enzyme

The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.

Control of phenylalanyl-tRNA synthetase genetic expression: Site-directed mutagenesis of the pheS, T operon regulatory region in vitro☆

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.

Switching from an induced-fit to a lock-and-key mechanism in an aminoacyl-tRNA synthetase with modified specificity.

Methionyl-tRNA synthetase (MetRS) specifically binds its methionine substrate in an induced-fit mechanism, with methionine binding causing large rearrangements. Mutated MetRS able to efficiently aminoacylate the methionine (Met) analog azidonorleucine (Anl) have been identified by saturation mutagenesis combined with in vivo screening procedures. Here, the crystal structure of such a mutated MetRS was determined in the apo form as well as complexed with Met or Anl (1.4 to 1.7 A resolution) to reveal the structural basis for the altered specificity. The mutations result in both the loss of important contacts with Met and the creation of new contacts with Anl, thereby explaining the specificity shift. Surprisingly, the conformation induced by Met binding in wild-type MetRS already occurs in the apo form of the mutant enzyme. Therefore, the mutations cause the enzyme to switch from an induced-fit mechanism to a lock-and-key one, thereby enhancing its catalytic efficiency.