C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction.

At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosome-dependent GTPase. The role of the eRF1-eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts of eRF1 (positions 1-280) and of eRF3 (positions 1477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478-530 and 628-637 of eRF3 and positions 281-305 and 411-415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411-415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase.