Kinetic analysis of interaction of eukaryotic release factor 3 with guanine nucleotides

Abstract

Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3·mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (Kd, ∼70 μM) is about 70-fold lower than for GDP (Kd, ∼ 1 μM) and both nucleotides dissociate rapidly from eRF3 (k-1mant-GDP ∼ 2.4 s-1; k-2mant-GTP ∼ 3.3 s -1). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg2+ concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3·GTP binding by two orders of magnitude (Kd ∼ 0.7 μM) due to lowering of the dissociation rate constant ∼24-fold (k-1mant-GTP ∼ 0.14 s-1). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1·eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.