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1.
J Biol Chem ; 276(23): 20064-8, 2001 Jun 08.
Article in English | MEDLINE | ID: mdl-11274157

ABSTRACT

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-tRNA(Met). 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 C(1)A(72) mismatch. In animal mitochondria, only one tRNA(Met) 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 tRNA(Met) 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 tRNA(Met) an A(1)U(72) motif.


Subject(s)
Hydroxymethyl and Formyl Transferases/metabolism , Mitochondria/enzymology , RNA, Transfer/metabolism , Amino Acid Sequence , Animals , Base Sequence , Catalysis , Cattle , Hydrolysis , Hydroxymethyl and Formyl Transferases/chemistry , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , RNA, Transfer/chemistry , Sequence Homology, Amino Acid
2.
EMBO J ; 17(23): 6819-26, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9843487

ABSTRACT

The crystal structure of Escherichia coli methionyl-tRNAfMet transformylase complexed with formyl-methionyl-tRNAfMet was solved at 2.8 A 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.


Subject(s)
Escherichia coli/enzymology , Escherichia coli/genetics , Hydroxymethyl and Formyl Transferases/chemistry , Nucleic Acid Conformation , Protein Conformation , RNA, Bacterial/chemistry , RNA, Transfer, Met/chemistry , Catalysis , Crystallography, X-Ray , Hydroxymethyl and Formyl Transferases/metabolism , RNA, Bacterial/metabolism , RNA, Transfer, Met/metabolism , RNA-Binding Proteins/chemistry
3.
Eur J Biochem ; 246(2): 539-47, 1997 Jun 01.
Article in English | MEDLINE | ID: mdl-9208948

ABSTRACT

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.


Subject(s)
Methionine-tRNA Ligase/chemistry , Methionine-tRNA Ligase/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Escherichia coli/enzymology , Geobacillus stearothermophilus/enzymology , Methionine/metabolism , Methionine-tRNA Ligase/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid , Zinc/metabolism
4.
Nucleic Acids Res ; 23(23): 4793-8, 1995 Dec 11.
Article in English | MEDLINE | ID: mdl-8532520

ABSTRACT

Methionyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family characterized both by a catalytic center built around a Rossmann Fold and by the presence of the two peptidic marker sequences HIGH and KMSKS. In this study, the role of the 21HLGH24 motif of Escherichia coli methionyl-tRNA synthetase was studied in a systematic fashion by site-directed mutagenesis. It is shown that the two histidine residues play a crucial role in the catalysis of the methionyl adenylate formation by participating in the stabilisation of the ATP phosphate chain during the transition state. Moreover, the results suggest the involvement of the epsilon-imino group of histidine 21 and of the delta-imino group of histidine 24. Notably, the substitution of either the leucine or the glycine residue of the HLGH motif by alanine had no effect on the catalysis. From the data and from other studies with class I aminoacyl-tRNA synthetases, concomitant positive contributions of the HIGH and KMSKS sequences to reach the transition state of aminoacyl adenylate formation can be envisaged.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Escherichia coli/enzymology , Methionine-tRNA Ligase/physiology , Methionine/analogs & derivatives , Adenosine Monophosphate/biosynthesis , Adenosine Triphosphate/metabolism , Methionine/biosynthesis , Methionine/metabolism , Methionine-tRNA Ligase/chemistry , Mutagenesis, Site-Directed , Peptide Fragments/metabolism , Structure-Activity Relationship
5.
J Mol Biol ; 233(4): 615-28, 1993 Oct 20.
Article in English | MEDLINE | ID: mdl-8411169

ABSTRACT

Escherichia coli methionyl-tRNA synthetase recognizes its cognate tRNAs according to the sequence of the CAU anticodon. In order to identify residues of methionyl-tRNA synthetase involved in tRNA anticodon recognition, enzyme variants created by cassette mutagenesis were genetically screened for their acquired ability to charge tRNA(mMet) derivatives with an ochre or an amber anticodon and, consequently, to cause the suppression of a stop codon in an indicator gene. The selected enzymes are called suppressors. Mutations were firstly directed towards the region of the synthetase encompassing residues 451 to 467. Several dozens of suppressor enzymes were compared. Statistical analysis of the mutations suggested that the substitution of an Asp side-chain at position 456 was sufficient to render possible the charging of the ochre or amber suppressor tRNAs. Point mutants at this position were therefore constructed. Their behaviour demonstrated that various tRNA(Met) derivatives having a non-Met anticodon could be aminoacylated in vitro provided only that the side-chain of residue 456 was no longer acidic. In turn, the Asp456 residue is not essential to the CAU anticodon recognition, since its substitution does not impair the aminoacylation of wild-type tRNA(Met). The analysis was enlarged to a second region from residue 437 to residue 454. The mutagenesis highlighted two other positions, one of which, Asn452, appeared involved in wild-type tRNA(Met) binding. The second position, Asp449, plays a role very similar to that of Asp456. It is concluded that both Asp449 and 456 behave as "antideterminants", contributing together to the rejection by the enzyme of tRNAs carrying non-Met anticodons. Finally, it is shown that the activities of some particular methionyl-tRNA synthetase variants, which have been made indifferent to the sequence of the anticodon of a tRNA(Met), are tightly dependent on the presence of the nucleotide determinants specific to the acceptor stem of tRNA(Met).


Subject(s)
Anticodon/metabolism , Escherichia coli/enzymology , Methionine-tRNA Ligase/metabolism , RNA, Transfer, Met/metabolism , Acylation , Amino Acid Sequence , Base Sequence , DNA, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Restriction Mapping , Substrate Specificity
6.
Nucleic Acids Res ; 19(13): 3673-81, 1991 Jul 11.
Article in English | MEDLINE | ID: mdl-1852609

ABSTRACT

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.


Subject(s)
Escherichia coli/enzymology , Geobacillus stearothermophilus/enzymology , Methionine-tRNA Ligase/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Codon/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Geobacillus stearothermophilus/genetics , Methionine-tRNA Ligase/metabolism , Molecular Sequence Data , Peptide Fragments , Sequence Homology, Nucleic Acid , Structure-Activity Relationship , Subtilisins/metabolism , Trypsin/metabolism
8.
Proc Natl Acad Sci U S A ; 88(1): 291-5, 1991 Jan 01.
Article in English | MEDLINE | ID: mdl-1986377

ABSTRACT

Accurate aminoacylation of a tRNA by Escherichia coli methionyl-tRNA synthetase (MTS) is specified by the CAU anticodon. A genetic screening procedure was designed to isolate MTS mutants able to aminoacylate a methionine amber tRNA (CUA anticodon). Selected suppressor MTS enzymes all possess one or several mutations in the vicinity of Trp-461, a residue that is the major contributor to the stability of complexes formed with tRNAs having the cognate CAU anticodon. Analysis of catalytic properties of purified suppressor enzymes shows that they have acquired an additional specificity toward the amber anticodon without complete disruption of the methionine anticodon site. It is concluded that both positive and negative discrimination toward the binding of tRNA anticodon sequences is restricted to a limited region of the synthetase, residues 451-467.


Subject(s)
Anticodon/metabolism , Escherichia coli/genetics , Genes, Bacterial , Methionine-tRNA Ligase/genetics , RNA, Transfer/metabolism , Suppression, Genetic , Anticodon/genetics , Escherichia coli/enzymology , Genetic Variation , Kinetics , Methionine-tRNA Ligase/metabolism , Mutagenesis, Site-Directed , RNA, Transfer/genetics , RNA, Transfer/isolation & purification
9.
Mol Gen Genet ; 223(1): 121-33, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2259334

ABSTRACT

The 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 tRNA(Met), 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.


Subject(s)
Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Methionine-tRNA Ligase/genetics , Transcription, Genetic , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/enzymology , Introns , Lac Operon , Methionine-tRNA Ligase/metabolism , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA, Messenger , RNA, Transfer, Met/metabolism , Restriction Mapping , Sequence Homology, Nucleic Acid , Terminator Regions, Genetic
10.
Biochimie ; 70(6): 773-82, 1988 Jun.
Article in English | MEDLINE | ID: mdl-3139093

ABSTRACT

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.


Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Chimera , Galactosidases/metabolism , Methionine-tRNA Ligase/genetics , Protein Engineering/methods , beta-Galactosidase/metabolism , Escherichia coli/genetics , Genetic Vectors , Mutation , Peptide Hydrolases/metabolism , Plasmids
11.
J Mol Biol ; 184(1): 31-44, 1985 Jul 05.
Article in English | MEDLINE | ID: mdl-3162032

ABSTRACT

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.


Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Gene Expression Regulation , Mutation , Operon , Phenylalanine-tRNA Ligase/genetics , Bacteriophage lambda/genetics , Base Sequence , DNA, Viral , Escherichia coli/enzymology , Escherichia coli/genetics , RNA, Messenger , RNA, Viral
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