title: C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids.
abstract: Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3. Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomyces cerevisiae eRF3 in addition to the EF-1alpha-like catalytic domain. By in vivo two-hybrid assay as well as by an in vitro pull-down analysis using purified proteins of S. pombe as well as of S. cerevisiae, eRF1 bound to the C-terminal one-third domain of eRF3, named eRF3C, but not to the N-terminal two-thirds, which was inconsistent with the previous report by Paushkin et al. (1997, Mol Cell Biol 17:2798-2805). The activity of S. pombe eRF3 in eRF1 binding was affected by Ala substitutions for the C-terminal residues conserved not only in eRF3s but also in elongation factors EF-Tu and EF-1alpha. These single mutational defects in the eRF1-eRF3 interaction became evident when either truncated protein eRF3C or C-terminally altered eRF1 proteins were used for the authentic protein, providing further support for the presence of a C-terminal interaction. Given that eRF3 is an EF-Tu/EF-1alpha homolog required for translation termination, the apparent dispensability of the N-terminal domain of eRF3 for binding to eRF1 is in contrast to importance, direct or indirect, in EF-Tu/EF-1alpha for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1alpha share some common amino acids for binding to eRF1 and aminoacyl-tRNA, respectively. These differences probably reflect the independence of eRF1 binding in relation to the G-domain function of eRF3 (i.e., probably uncoupled with GTP hydrolysis), whereas aminoacyl-tRNA binding depends on that of EF-Tu/EF-1alpha(i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.
authors.affiliation: Department of Tumor Biology, The Institute of Medical Science, The University of Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ebihara
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism*; D011328:Prions; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: C489002:ETF1 protein, human; D005656:Fungal Proteins; D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C087971:peptide-chain-release factor 3; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate