The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two‐hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C‐terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon‐specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA‐containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti‐suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C‐terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.

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Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRFI), which has a stop-codon-dependent peptidyl-release activity. Using a conditionallethal mutant allele of SUP45 (sup4.5-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45-2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37°C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature. These data demonstrate that yeast eRFl plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues.Keywords: Saccharomyces cerevisiae ; translation termination ; eukaryotic release factor ; SUP45 ; conditional-lethal mutant.Allosuppressor (sal) mutants of the yeast Saccharomyces cerevisiae were originally identified by their ability to enhance the efficiency of the weak ochre suppressor tRNA encoded by the SUQ.5 gene (Cox, 1977). Alleles of two of these sal genes (sa13 and sa14) show an omnipotent suppressor phenotype (i.e. suppression of a nonsense mutation in the absence of a mutant suppressor tRNA) and are allelic to the SUP35 and SUP45 genes, respectively (Stansfield and Tuite, 1994). These phenotypes are consistent with the mutants having a defect in translation termination since the efficiency of nonsense suppression reflects the ability of a suppressor tRNA to compete with the endogenous termination mechanism for a stop codon (in the case of the SUQ5 suppressor, UAA) positioned at the ribosomal A site. A defect in the termination mechanism will be expected to increase the efficiency with which the suppressor tRNA is able to recognise the cognate codon.The product of the SUP45 gene (originally designated Sup45p) is a 438-amino-acid polypeptide (Breining and Piepersberg, 1986), which is tightly associated with the ribosome and is present in exponentially growing cells at a stoichiometry of approximately 1 mol Sup45p/20 mol ribosomes (Stansfield et al., 1992 with an AUG codon followed by a tetranucleotide stop signal: Sup45p-dependent peptidyl-releaqe activity stimulates release of 35S-labelled methionine from methionyl-tRNA on the ribosome (Frolova et al., 1994). This in vitm release-factor act...

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