The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast.
Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3. The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis. In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1. The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35). All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids. To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae. These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.
Department of Tumor Biology, The Institute of Medical Science, The University of Tokyo, Japan.
K
K
Ito
K
K
Ebihara
Y
Y
Nakamura
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010443:Peptide Chain Termination, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D010454:Peptide Termination Factors; C087971:peptide-chain-release factor 3; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu