A CONDITIONAL-LETHAL TRANSLATION TERMINATION DEFECT IN A SUP45 MUTANTOF THE YEAST SACCHAROMYCES-CEREVISIAE

Genetic studies have indicated that the product of the yeast SUP45 gen e encodes a component of the translational-termination machinery. In h igher eukaryotes, genes similar to SUP45 encode eukaryote release fact or 1 (eRF1), which has a stop-codon-dependent peptidyl-release activit y. Using a conditional lethal mutant allele of SUP45 (sup45-2) and a c ombination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for t ranslation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show th at a translating lysate from a sup45-2 mutant strain exhibits a termin ation defect when heated for short periods to greater than the non-per missive temperature (37 degrees C). This defect can be complemented wi th a purified preparation of Sup45p (eRF1) expressed in Eschericha col i, The termination defect in this strain appears to be due to an inabi lity of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the r ibosome at the non-permissive temperature. Cell-free translation lysat es from the sup45-2 strain do not show a defect in sense-codon transla tion at the non-permissive temperature. These data demonstrate that ye ast eRF1 plays a role in translation termination and is functionally e quivalent to its higher eukaryotic homologues.