Termination of translation in higher organisms is a GTP‐dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35‐like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C‐domain of the X.laevis eRF3 protein is highly conserved with other Sup35‐like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.
Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N؉M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 ( In general, termination of protein synthesis occurs when the ribosome elongation machinery encounters an in-frame termination codon on the mRNA. In eukaryotes two release factors have been identified, eukaryotic release factor 1 (eRF1), which recognizes all three stop codons, and eRF3, a GTPase that binds to eRF1 and stimulates its release activity in vitro (35,109). The eRF1 protein has a structure mimicking that of a tRNA molecule. It recognizes the stop codon in the A site of the ribosome and catalyzes the hydrolysis of the peptidyltRNA bond (88).In Saccharomyces cerevisiae eRF1 and eRF3 termination factors are encoded by the essential genes SUP45 and SUP35, respectively (10,43,53,60,105). Mutations in either of these genes give the same pleiotropic phenotypes which were selected as omnipotent nonsense suppressors (for a review see reference 49). Moreover, overexpression of both eRF1 and eRF3 is required to enhance the efficiency of termination in yeast (90). In higher eukaryotes, overproduction of eRF1 alone is sufficient to compete with a suppressor tRNA (62). Either Xenopus laevis or human eRF1 alone was also shown previously to have an antisuppressor effect against a suppressor tRNA in the reticulocyte lysate translation system (28). In vitro the eRF1 of higher eukaryotes has a release activity and does not need any other factor (28, 35), and eRF3 by itself binds GTP, but GTPase activity requires the presence of both eRF1 and ribosomes (36). It has been shown previously that eRF1 and eRF3 interact, suggesting that they form a functional complex (70,90,99,109 . Yeast eRF3 consists of an N-terminal prionforming domain (PrD), a charged M (middle) domain of unknown function, and a C-terminal domain that provides the essential translation termination activity (96,97,105). Recently, the minimum length of PrD was defined as amino acids (aa) 1 to 97 (76). It was shown previously that Hsp104 protein is required for formation and maintenance of [PSI ϩ ] aggregates of eRF3: its overproduction or inactivation cures cells of [PSI ϩ ] (14). The eRF3 family includes proteins from yeasts, humans, X. laevis, and other species that are strongly conserved in the C-terminal region, which has a significant homology with the translation elongation factor eEF1A (47,60,109). In contrast to the C-terminal part, the N-terminal region of eRF3 is ...
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