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-1a-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-1a. 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-1a 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-1a for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1a 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-1a (i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.

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.…”

Suppression of eukaryotic translation termination by selected RNAs

“…In contrast to eRF1, Class 2 RFs (RF3 and eRF3) do not act as ribonucleoproteins+ Instead they are GTPbinding proteins (Grentzmann et al+, 1994;Mikuni et al+, 1994;Zhouravleva et al+, 1995) that hydrolyze the triphosphate when bound to the ribosome (Frolova et al+, 1996;Freistroffer et al+, 1997;Grentzmann et al+, 1998;Pel et al+, 1998)+ Interaction with eRF1 is essential for eRF3 GTPase, via contacts between their C-termini (Ebihara & Nakamura, 1999;Merkulova et al+, 1999)+ Such eRF1•eRF3 complexes are evident both in vivo and in vitro (Stansfield et al+, 1995;Zhouravleva et al+, 1995;Paushkin et al+, 1997)+ Thus, although eRF3 is probably not a natural RNA-binding protein, we still expect surfaces with potential RNA affinity on the protein+ RNAs with high affinities can be selected to virtually every protein, even toward peptide domains that lack natural sites for ribonucleotides or other polyanions (Gold et al+, 1995)+ Our selection yielded RNAs that definitely contact eRF3+ These are the Class II RNAs like RNA 27 (Figs+ 1 and 3) that have a unique multihelix-junction structure and complementary affinities for eRF1 and eRF3 alone (Fig+ 2)+ We suggest that these RNAs actually bridge the eRF1•eRF3 interface in the rightward (C domain; carboxyl-terminal) domain of the eRF1 structure (Song et al+, 2000)+ Such a distinct site would be consistent with the observed unique primary and secondary structures, which differ from Class I and eRF1 aptamers (Figs+ 1 and 3)+ However, Class II RNAs do not inhibit RF activity (Table 2)+ Because Class II RNAs were selected against the heterodimer, their binding may be consistent with a functional eRF1•eRF3 interface+ Thus, they may allow functional interactions between the RF proteins, as well as continued release function by the relatively distant NM domain+…”