METHIONYL-TRANSFER RNA-SYNTHETASE GENE FROM AN EXTREME THERMOPHILE, THERMUS-THERMOPHILUS HB8 - MOLECULAR-CLONING, PRIMARY-STRUCTURE ANALYSIS, EXPRESSION IN ESCHERICHIA-COLI, AND SITE-DIRECTED MUTAGENESIS

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 MetRS 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 MetRS. 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).