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

Abstract

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 ribosomedependent GTPase. The role of the eRF1ceRF3 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 Nterminal parts of eRF1 (positions 1^280) and of eRF3 (positions 1^477) 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 4114 15 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 Cterminally truncated eRF3 is inactive as a GTPase.z 1999 Federation of European Biochemical Societies.

C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids

“…Truncated eRF1 proteins and their binding to the C-terminal domain of S. pombe eRF3+ The activity of the eRF3C segment (amino acid positions 482-662) for binding to different truncated eRF1 polypeptides was examined by an in vivo two-hybrid system as well as by an in vitro pull-down analysis, as shown in Figure 4, and the data are summarized+ ϩ: binding, 6: weak binding; Ϫ: no binding+ The number refers to the amino acid position from the translation start site+ Sequence motifs homologous to domains III (acceptor stem mimicry), IV (anticodon helix mimicry), and V (T stem mimicry) of elongation factor EF-G are assigned (Ito et al+, 1996(Ito et al+, , 1998a)+ interaction of the C-terminal one-third domain of eRF3 per se with eRF1+ The present study has also confirmed the importance of the C-terminal acidic amino acid stretch of S. pombe eRF1 for binding to eRF3 (Ito et al+, 1998a)+ Following near completion of the revised manuscript after submission, we became aware of a recent study that warrants mention+ Merkulova et al+ (1999) have reported the similar C-terminal domain activity of human eRF3 for binding to eRF1+…”

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

“…The C-terminal region of eRF3 has been found to be the primary binding site for eRF1 (Ebihara and Nakamura 1999;Merkulova et al 1999). In this region, the highly conserved GRFTLRD motif plays a crucial role in mediating eRF1 binding (Kong et al 2004).…”

“…Recent data suggest that GTP hydrolysis by eRF3 couples stop codon recognition by eRF1 with peptidyl-tRNA hydrolysis by the peptidyl transferase center (Salas-Marco and Bedwell 2004; Alkalaeva et al 2006). eRF1 and eRF3 form a stable complex through interaction of their C-terminal domains (Ito et al 1998;Ebihara and Nakamura 1999;Merkulova et al 1999). Studies of the interaction of guanine nucleotides with eRF3 alone and with the eRF3deRF1 complex show that GTP and eRF1 bind to eRF3 with strong positive cooperativity (Hauryliuk et al 2006;Pisareva et al 2006) and that the stimulatory effect of eRF1 on binding of eRF3 to GTP strongly depends on the presence of Mg 2+ .…”

Eukaryotic release factors (eRFs) history

“…It was also shown that the products of SUP45 (eRF1) and SUP35 (eRF3) interact in vitro (Stansfield et al 1995b;Zhouravleva et al, 1995). This interaction is mediated by the C-terminal part of eRF1, although there are some discrepancies about the precise localization of the region of eRF1 that interacts with eRF3 Ebihara and Nakamura, 1999;Eurwilaichitr et al, 1999;Merkulova et al, 1999). The deletion of the C-terminal 19 amino acids abolishes the interaction with eRF3 and causes an enhancement of nonsense suppression.…”