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 (K(d), approximately 70 microM) is about 70-fold lower than for GDP (K(d), approximately 1 microM) and both nucleotides dissociate rapidly from eRF3 (k(-1)(mant-GDP) approximately 2.4 s(-1); k(-2)(mant-GTP) approximately 3.3 s(-1)). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg(2+) concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3.GTP binding by two orders of magnitude (K(d) approximately 0.7 microM) due to lowering of the dissociation rate constant approximately 24-fold (k(-1)(mant-GTP) approximately 0.14s(-1) approximately 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 x 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.
Highlights
Our studies revealed that mutual interdependence of eRF1 and eRF3 in the termination process involves stimulation of GTP hydrolysis by eRF3 on the ribosome and peptide release induced by eRF1, and stimulation by eRF1 of guanine nucleotide binding to eRF3
To investigate guanine nucleotide binding to eRF3, we used recombinant human eRF3aC lacking the N-terminal 138 amino acids, which are dispensable for the eRF3 GTPase activity and for its stimulation of eRF1 release activity [12]
In this report we describe a kinetic and thermodynamic analysis of the interaction of eRF3 with guanine nucleotides in the absence and in the presence of eRF1 and 80 S ribosomes
Summary
Chemicals—The fluorescent GDP/GTP derivatives mantGDP and mant-GTP were from Jena Bioscience (Jena, Germany), a 100 mM GTP solution was from GE Biosciences (Piscataway, NJ) and a 100 mM GDP solution was from Roche Applied Science. The chase of mant-GTP by GDP from eRF3aC1⁄7eRF1 in the presence of 2.5 mM Mg2ϩ was measured using 0.8 M eRF3aC, 8 M eRF1, 3 M mant-GTP, and increasing concentrations of unlabeled GDP. Where P is eRF3aC or the eRF3aC1⁄7eRF1 complex; A is mantGTP or mant-GDP; I is GTP or GDP; KA and KI are equilibrium dissociation constants of A and I, respectively In this case the fluorescence of a mant-guanine nucleotide observed in the presence of a competitor unlabeled guanine nucleotide, F(I), can be described by the following Equation 6, FIϭ Fmax ϫA/͓͑Aϩ KA1 ϩI/KI͒ ϩ Fmin (Eq 6). Blank values resulting from the presence of 3.2% Pi in [␥-32P]GTP preparations were determined in the absence of eRF3 and subtracted from all measurements
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
