We describe the development of a novel plasmid-based assay for measuring the in vivo frequency of misincorporation of amino acids into polypeptide chains in the yeast Saccharomyces cerevisiae. The assay is based upon the measurement of the catalytic activity of an active site mutant of type III chloramphenicol acetyl transferase (CATIII) expressed in S. cerevisiae. A His195(CAC)-->Tyr195(UAC) mutant of CATIII is completely inactive, but catalytic activity can be restored by misincorporation of histidine at the mutant UAC codon. The average error frequency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0. 5x10(-5) and this frequency was increased some 50-fold by growth in the presence of paromomycin, a known translational-error-inducing antibiotic. A detectable frequency of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2x10(-5), but in contrast to the Tyr195-->His195 misincorporation event, the frequency of histidine misincorporation at Ala195 GCU was not increased by paromomycin, inferring that this error did not result from miscognate codon-anticodon interaction. The His195 to Tyr195 missense error assay was used to demonstrate increased frequencies of missense error at codon 195 in SUP44 and SUP46 mutants. These two mutants have previously been shown to exhibit a translation termination error phenotype and the sup44+ and sup46+ genes encode the yeast ribosomal proteins S4 and S9, respectively. These data represent the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arising from non-cognate codon-anticodon interactions.

• PMID: 9733638
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Stansfield I, Jones KM, Herbert P, Lewendon A, Shaw WV, Tuite MF

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