Identification of translational release factor eRF1a binding sites on eRF3 in Euplotes octocarinatus

Abstract

Translation termination in eukaryotes is mediated by two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, while eRF3 is a ribosome-dependent and eRF1-dependent GTPase. eRF1 forms a stable complex with eRF3 in vivo and in vitro. In the present study, a variety of truncated forms of Euplotes octocarinatus eRF3 were created, and systematic analysis of the interaction between E. octocarinatus eRF1a and these eRF3 mutants was performed by employing both in vivo a yeast two-hybrid assay and in vitro a pull-down assay. The results demonstrated that a short portion of the C-terminal domain of eRF3 is sufficient for eRF1a binding in E. octocarinatus. Specifically, the eRF1a-binding sites on eRF3 are located at a region containing amino acid residues 640-723 in E. octocarinatus eRF3. Amino acid sequence analysis of eRF3 from E. octocarinatus, humans and yeast showed that the eRF1a binding domain on E. octocarinatus eRF3 was similar to that of yeast eRF3 but different from that of human eRF3.