SACCHAROMYCES-CEREVISIAE CYTOPLASMIC TYROSYL-TRANSFER RNA-SYNTHETASE GENE - ISOLATION BY COMPLEMENTATION OF A MUTANT ESCHERICHIA-COLI SUPPRESSOR TRANSFER-RNA DEFECTIVE IN AMINOACYLATION AND SEQUENCE-ANALYSIS

Exploiting differences in tRNA recognition between prokaryotic and euk aryotic tyrosyl-tRNA synthetases (TyrRSs), we have isolated the gene f or the cytoplasmic TyrRS of Saccharomyces cerevisiae by functional com plementation in Escherichia coli of a mutant E. coli tRNA. The tRNA, d erived from the E. coli initiator tRNA with changes to allow suppressi on of amber termination codons, is poorly aminoacylated in E. coli and hence, is only a weak amber suppressor. The same tRNA functions as a good suppressor in S. cerevisiae and is aminoacylated with tyrosine by yeast extracts. We expressed a yeast cDNA library in an E. coli strai n carrying the mutant tRNA gene and several genes with amber mutations . cDNA clones were isolated which increased suppression and levels of amino-acylation of the mutant tRNA. Characterization of the gene ident ified a methionine-initiated open reading frame encoding a protein of 394 amino acids. Expression of this protein in E. coli demonstrated th at tyrosine was incorporated during suppression and that yeast cytopla smic TyrRS activity was produced. Yeast cytoplasmic TyrRS has sequence s typical of class I aminoacyl-tRNA synthetases, but only weak overall sequence similarity to the corresponding eubacterial and mitochondria l TyrRSs. However, many of the residues known to line the tyrosyl-aden ylate-binding pocket of the Bacillus stearothermophilus enzyme can be aligned in the yeast sequence. These include the aspartic acid and tyr osine residues thought to contact the tyrosine side chain to provide s ubstrate specificity.