Timing of GTP binding and hydrolysis by translation termination factor RF3

1812–1820 Nucleic Acids Research, 2014, Vol. 42, No. 3 doi:10.1093/nar/gkt1095 Published online 8 November 2013 Timing of GTP binding and hydrolysis by translation termination factor RF3 Frank Peske, Stephan Kuhlenkoetter, Marina V. Rodnina and Wolfgang Wintermeyer* Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany Received July 18, 2013; Revised October 17, 2013; Accepted October 18, 2013 Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock. INTRODUCTION Important steps of translation on the ribosome in bacteria are controlled by guanosine triphosphatases (GTPases), including initiation factor IF2, elongation factors EF-Tu and EF-G and release factor RF3. These GTPases share the same binding site on the ribosome and are activated on interaction with the ribosome (1). In the termination phase of protein synthesis, release factor 1 or 2 (RF1/2) recognizes stop codons on the messenger RNA (mRNA) in the A site (2). On binding of RF1/2 to a stop codon, the universally conserved GGQ motif of RF1/2 reaches into the peptidyl transferase center (3) and promotes peptidyltRNA hydrolysis (4–7). RF3 accelerates the dissociation of RF1/2 from the ribosome after peptide release (8). Another function of RF3 has been observed in quality control during translation elongation where RF3 stimulated the hydrolysis of erroneous peptidyl-tRNA by RF1/2 (9,10). Crystal structures of ribosome complexes with RF3 and the non-hydrolyzable GTP analog GDPNP (11,12) indicate that in these complexes the ribosome is present in the hybrid/rotated state, whereas the pre- or post-termination ribosome complexes with RF1/2 assume the classic non-rotated state (13–16). The structures of ribosome–RF3 complexes indicate that RF1/2 binding would be destabilized in the hybrid/rotated state due to steric clashes, explaining why RF3 promotes the release of RF1/2. The role of GTP binding and hydrolysis for the function of RF3 has not been fully clarified yet. A model was proposed (17–19), in which RF3–guanosine diphosphate (GDP), but not RF3–GTP, binds to the pre-termination complex (PreTC), i.e. the ribosome with peptidyl-tRNA in the P site and RF1/2 bound to a termination codon in the A site. According to that model, ribosome binding of RF3 accelerates GDP dissociation and re-binding, but GTP can only bind following the RF1/2-induced peptide release forming the post-termination complex (PostTC). Further, GTP binding to RF3 results in the release of RF1/2 from the ribosome, which, in turn, induces GTP hydrolysis. Owing to the low stability of RF3–GDP on the ribosome, RF3–GDP dissociates from the ribosome, completing the functional cycle. Important features of that model were based on nonequilibrium measurements of nucleotide binding to RF3, using a nitrocellulose filter binding assay, which has proven unreliable for kinetically unstable nucleotide complexes of translation factors (20). Furthermore, except for a few stopped-flow data on the dissociation of *To whom correspondence should be addressed. Tel: +49 551 2012902; Fax: +49 551 2012905; Email: The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Nucleic Acids Research, 2014, Vol. 42, No. 3 1813 GDP from RF3 on the ribosome (21), information from time-resolved experiments is lacking so far. The aim of the present article was to re-examine the effect of RF1/2 on GDP–GTP exchange on RF3, to measure guanine nucleotide affinities for binding to free and ribosome-bound RF3 at equilibrium and to determine the timing of peptide release and GTP hydrolysis during termination. MATERIALS AND METHODS Materials Rapid kinetics Rapid kinetic experiments were performed on an SX20MV stopped-flow apparatus (Applied Photophysics, Leatherhead, UK). Experiments were performed by rapidly mixing equal volumes (60 ml) of reactants at 37 C. RF3–guanine nucleotide complex formation or dissociation was monitored by the fluorescence of mantGDP, mantGTP or mantGDPNP, which was excited by FRET from tryptophan in RF3. The excitation wavelength was Equilibrium titrations RF3–GDP (0.5–4 mM) was mixed with mantGTP, mantGDP or mantGDPNP as indicated (Figure 3A). PreTC or PostTC (0.05 mM) was mixed with RF2(GGA) (2 mM) and RF3–mantGTP/GDP was prepared using a 2-fold excess as indicated (Figure 3). For competition titrations with unlabeled guanine nucleotides (Figure 3B), the RF3–mantGDP complex was formed (40 mM RF3, 400 mM mantGDP) and purified on a NAP5 column. Fluorescence titrations were performed in a FluoroLog 3 spectrofluorimeter (Horiba Scientific; Edison, NJ, USA). The fluorescence of the mant group was excited by FRET from tryptophan in RF3. The excitation wavelength was 290 nm, and the emission was measured at 445 nm. The fluorescence change due to complex formation,
F, was determined by subtracting the signals obtained in a control titration of mant-labeled nucleotide in buffer without RF3 from the signals obtained in the presence of RF3 and its complex partners, if present. Difference plots were evaluated using the following quadratic equation, accounting for the concentration change of added ligand due to complex formation,
F ¼ 0:5  Bmax =P n h io 2 Kapp +P+X  sqrt Kapp +P+X 4  P  X , where Bmax represents the amplitude, P the total concentration of RF3, X the total concentration of nucleoti (...truncated)