MISSENSE TRANSLATION ERRORS IN SACCHAROMYCES-CEREVISIAE

We describe the development of a novel plasmid-based assay for measuri ng the in vivo frequency of misincorporation of amino acids into polyp eptide chains in the yeast Saccharomyces cerevisiae. The assay is base d upon the measurement of the catalytic activity of an active site mut ant of type III chloramphenicol acetyl transferase (CAT(III)) expresse d in S. cerevisiae. A His195(CAC) --> Tyr195(UAC) mutant of CAT(III) i s completely inactive, but catalytic activity can be restored by misin corporation of histidine at the mutant UAC codon. The average error fr equency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0.5 x 10(-5) and this frequen cy was increased some 50-fold by growth in the presence of paromomycin , a known translational-error-inducing antibiotic. A detectable freque ncy of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2 x 10(-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 erro r did not result from miscognate codon-anticodon interaction. The His1 95 to Tyr195 missense error assay was used to demonstrate increased fr equencies of missense error at codon 195 in SUP44 and SUP46 mutants. T hese two mutants have previously been shown to exhibit a translation t ermination error phenotype and the sup44(+) and sup46(+) genes encode the yeast ribosomal proteins S4 and S9, respectively. These data repre sent the first accurate in vivo measurement of a specific mistranslati on event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arisi ng from non-cognate codon-anticodon interactions. (C) 1998 Academic Pr ess.