Abstract
The gene for the methionyl-tRNA synthetase (MetRS) from an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. By expression of the T. thermophilus MetRS gene in Escherichia coli cells, thermostable MetRS was overproduced and purified to homogeneity by heat treatment and one-step column chromatography. The amino acid sequence of T. thermophilus MetRS showed low identities (approximately 25%) with those of MetRSs from E. coli, and cytoplasm and mitochondria of Saccharomyces cerevisiae. However, the amino acid residues in the binding sites for ATP and the anticodon and the 3' terminus of tRNA(Met) are highly conserved among the four MetRSs. T. thermophilus MetRS has a zinc finger-like sequence with all the three cysteine residues and a histidine residue. By site-directed mutagenesis of one of the cysteine residues (Cys127) of T. thermophilus MetRS, the SH group was found to be important for methionyl-tRNA synthesis. Just upstream of the structural gene for T. thermophilus MetRS there is a short open reading frame which codes for a methionine-rich peptide and is partly overlapped with an alternative terminator/antiterminator structure, suggesting that transcription of this gene is regulated by attenuation. Further upstream a region contains a nucleotide sequence homologous to that of the 5' half of T. thermophilus initiator tRNA(Met).
Highlights
Further,theamino acidresidues aroundthe tRNA-binding site of E. coli methionyl-tRNA synthetase (MetRS) have been identified by cross-linking experiments[10,11,12,13,14,15,16]
The open reading frame of the metS gene was composed of 1848 bp, from which the sequence of 616 amino acid residues comprising the subunit of T.thermophilus MetRS was deduced (Fig. 2)
From x-ray crystallographic studies, a trypsin-modified E. coli MetRS that lacks the COOH-terminal domain has been reported to consist of two domains [19],which probably correspond to domains T1 and T2,respectively, of T. thermophilus MetRS
Summary
Bacterial Strains-T. thermophilus strain HB8 (ATCC 27634) [23] was a generous gift from Dr.T. Molecular Cloning of the T. thermophilus metS Gene-On. Chromosomal DNA of T. thermophilus was prepared by the method the basis of the NH2-terminal amino acid sequences of doof Marmur [32]. Thermophilus chromosomal DNA digested with several restriction enzymes Both of 23-mer and 29-mer probes hybridized to thesame KpnI fragment of about 1.5 kb (data notshown). Oligonucleotide-directedMutagenesis of the T. thermophilus metS Gene-Plasmid pUC118/TMTS-2.2 (Fig. 1) was digested with Sac and BamHIor with KpnI,and the resultant1.1-kbSucIIBamHI deduced from the nucleotide sequence included the NH2 termini of domains T2 and T3 of the MetRS, indicating that this fragment was a part of the T. thermophilus metS gene. The open reading frame of the metS gene was composed of 1848 bp, from which the sequence of 616 amino acid residues comprising the subunit of T.thermophilus MetRS was deduced (Fig. 2).
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