Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae

We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells.Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.Termination of protein synthesis occurs when the ribosome elongation machinery encounters an in-frame termination (stop) codon, either UAG, UGA or UAA, in the mRNA. This stop codon located in the A-site of the ribosome is recognized by a release factor (RF1/RF2 in prokaryotes and eRF1 in eukaryotes), which triggers release of the nascent peptide from the ribosome. Termination efficiency is enhanced by the GTPase release factor, RF3 in prokaryotes and eRF3 in eukaryotes [reviewed in [1-4]].Release factor eRF1 recognizes all three stop codons [5], in contrast to prokaryotes where RF1 catalyses translation termination at UAG and UAA codons, and RF2 at UGA and UAA codons [6]. All these proteins are encoded by essential genes: prfA (for RF1), prfB (for RF2) in bacteria [7-9] and SUP45 (for eRF1) in S. cerevisiae [10,11]. Although the sequences of prokaryotic and eukaryotic release factors differ significantly [see [12] for review], a "tRNA-mimicry" model generalizing the mechanisms of prokaryotic and eukaryotic translation termination, was proposed [13]. Peptide determinants of RF1 and RF2 involve