The sequence of a 5-kilobase DNA insert containing the structural gene for yeast cytoplasmic methionyl-tRNA synthetase has been determined and a unique open reading frame of 2,253 nucleotides encoding a polypeptide chain of 751 amino acids (Mr, 85,5W) has been characterized. The data obtained on the purified enzyme (subunit size, amino acid composition, and COOH-terminal sequence) are consistent with the gene structure. The protein sequence deduced from the nucleotide sequence reveals no obvious internal repeats. This protein sequence shows a high degree of homology with that of Eacherichia coli methionyltRNA synthetase within a region that forms the putative methionyl adenylate binding site. This strongly suggests that both proteins derive from a common ancestor.Aminoacyl-tRNA synthetases play a crucial role in protein synthesis because they catalyze the specific attachment of amino acids to their cognate tRNAs. Knowledge of their primary structure is one of the prerequisites for the complete understanding of the structure-function relationship. So far, the sequence of only one aminoacyl-tRNA synthetase has been completely determined by using classical protein sequence analysis techniques (1). However, the cloning of a number of aminoacyltRNA synthetases genes has allowed derivation of the primary structure of the corresponding enzyme from the DNA sequence-i.e., the alanyl-(2), tryptophanyl-(3), glutaminyl-(4), and methionyl-(5) tRNA synthetases from Escherichia coli.In our laboratory, a yeast mutant strain lacking functional cytoplasmic methionyl-tRNA synthetase was available. It was complemented with a plasmid pool containing random fragments of wild-type yeast genomic DNA obtained by a partial Sau3A digestion (6). It was thus possible to isolate a 5. 1-kilobase piece of DNA containing the methionyl-tRNA synthetase gene (MES1). The isolated gene product is a monomer (Mr, 80,000). However, in crude extracts from a wild-type strain the enzyme behaves as a dimer (Mr, 2 x 80,000). Because enzyme purification always led to a monomeric species with no detectable variation of Mr, the existence of a dimeric structure for native methionyl-tRNA synthetase is by no means proven (unpublished data).In this paper we report the complete nucleotide sequence of the DNA insert containing the structural gene. Indeed, a unique open reading frame whose length corresponds exactly to that expected for the above protein size could be characterized.MATERIALS AND METHODS DNA Sequence Analysis. DNA sequence analysis was carried out by the chain termination method of Sanger et al. (7).Fragments of the cloned DNA were subeloned into the bacteriophage M13mp7 (8) and analyzed by using the primer synthesized by R. Crea and purchased from P-L Biochemicals.Enzymes and Chemicals. Most restriction enzymes and T4 DNA ligase were obtained from New England BioLabs. The Klenow E. coli polymerase and nuclease SI were from Boehringer Mannheim.All chemicals were from Merck (Darmstadt, Federal Republic of Germany) and were analytical grade.
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