Termination of translation in eukaryotes is mediated by the quaternary eRF1•eRF3•GTP•Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

Abstract

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (Kd = 1.9 μM) and this interaction depends only minimally on the Mg2+ concentration; (ii) GTP binds to eRF3 (Kd = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.

Cryo-EM structure of the mammalian eukaryotic release factor eRF1–eRF3-associated termination complex

“…The higher-resolution (∼9.5 Å) cryo-EM structure of the yeast 80S-Dom34-Hbs1-GMPPNP complex revealed a homologous interaction between the middle domain of Dom34 (which is structurally homologous to domain M of eRF1 except that it lacks the GGQ motif) and the G-domain of Hbs1, with the positively charged loop β10-α3 loop of Dom34 directly contacting the switch-1 region of Hbs1 (29). The similarities in overall architecture of the eRF1-eRF3-and Dom34-Hbs1-bound ribosomal complexes in a pre-GTP hydrolysis state suggest a conserved mechanism of ribosome binding and GTP hydrolysis and are consistent with the essential roles of eRF1 and Dom34 in promoting GTP binding and hydrolysis by eRF3 and Hbs1, respectively (5,(18)(19)(20)(21)(22)36).…”

“…Association with eRF1 specifically stabilizes binding of GTP to eRF3 by two orders of magnitude (18)(19)(20) so that eRF1 and eRF3 form a stable eRF1-eRF3-GTP ternary complex. eRF1 is also strictly required for stimulation of eRF3's ribosomedependent GTPase activity (21).…”

S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

“…Isothermal Titration Calorimetry-The thermodynamic parameters of adenine nucleotide binding to rabbit Na,K-ATPase were measured using a MicroCal iTC200 instrument (MicroCal, Northampton, MA), as described elsewhere (23). Experiments with nonglutathionylated (dithiothreitol (DTT), 100 M) and glutathionylated (GSSG, 1 mM) Na,K-ATPase were carried out at 25°C in imidazole buffer containing 25 mM imidazole, 1 mM EDTA, 250 mM sucrose, pH 7.5.…”

Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system

“…Briefly, the binding reaction mixtures containing 5 0 -[32P] labeled 2 nM rU 12 , and 0-500 nM NS5A protein were kept on ice for 30 min and then filtered through the membranes which were dried on air. The radioactivity was visualized as described above.…”

“…Experiments were carried out at 25°C in buffer C (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM EDTA, and 5 mM b-mercaptoethanol) similarly to [12]. Briefly, aliquots (10 ll) of NS5A protein were injected into the 1.42 ml cell containing the NS5B solution.…”