Saccharomyces cerevisiae cytoplasmic tyrosyl-tRNA synthetase gene. Isolation by complementation of a mutant Escherichia coli suppressor tRNA defective in aminoacylation and sequence analysis.
Exploiting differences in tRNA recognition between prokaryotic and eukaryotic tyrosyl-tRNA synthetases (TyrRSs), we have isolated the gene for the cytoplasmic TyrRS of Saccharomyces cerevisiae by functional complementation in Escherichia coli of a mutant E. coli tRNA. The tRNA, derived from the E. coli initiator tRNA with changes to allow suppression 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 strain carrying the mutant tRNA gene and several genes with amber mutations. cDNA clones were isolated which increased suppression and levels of aminoacylation of the mutant tRNA. Characterization of the gene identified a methionine-initiated open reading frame encoding a protein of 394 amino acids. Expression of this protein in E. coli demonstrated that tyrosine was incorporated during suppression and that yeast cytoplasmic TyrRS activity was produced. Yeast cytoplasmic TyrRS has sequences typical of class I aminoacyl-tRNA synthetases, but only weak overall sequence similarity to the corresponding eubacterial and mitochondrial TyrRSs. However, many of the residues known to line the tyrosyl-adenylate-binding pocket of the Bacillus stearothermophilus enzyme can be aligned in the yeast sequence. These include the aspartic acid and tyrosine residues thought to contact the tyrosine side chain to provide substrate specificity.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
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RajBhandary
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004271:DNA, Fungal; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D012358:RNA, Transfer, Met; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004271:DNA, Fungal; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D012365:RNA, Transfer, Tyr; C024988:tRNA, formylmethionine-; D014448:Tyrosine-tRNA Ligase