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-tR NA is jointly catalysed at the ribosome by the termination codon and the po lypeptide release factor (eRF1 in eukaryotes), eRF1 forms in vivo and in vi tro a stable complex with release factor eRF3, an eRF1-dependent and riboso me-dependent GTPase. The role of the eRF1(.)eRF3 complex in translation rem ains 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 1-477) are either not involved or non-essential for binding. Two regions i n each factor are critical for mutual binding: positions 478-530 and 628-63 7 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-t erminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPas e. (C) 1999 Federation of European Biochemical Societies.