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
Cm. Chow et Ul. Rajbhandary, 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, The Journal of biological chemistry, 268(17), 1993, pp. 2855-2863
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.