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Escherichia Coli Release Factor 3: Resolving the Paradox of a Typical G Protein Structure and Atypical Function with Guanine Nucleotides

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Escherichia Coli Release Factor 3: Resolving the Paradox of a Typical G Protein Structure and Atypical Function with Guanine Nucleotides Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press RNA (1998), 4:47–54. Cambridge University Press. Printed in the USA. Copyright © 1998 RNA Society. Escherichia coli release factor 3: Resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides HERMAN J. PEL,1* JOHN G. MOFFAT,1* KOICHI ITO,2 YOSHIKAZU NAKAMURA,2 and WARREN P. TATE1 1Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand 2Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Japan ABSTRACT Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3{GDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3{GDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome- mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction. Keywords: G protein; protein synthesis; ribosome; RF3; translational termination INTRODUCTION ity was inhibited by several forms of guanine nucleo- tides; GDP, GTP, and nonhydrolysable GTP analogues The termination step of protein synthesis requires rec- abolished the effect of the nucleotide-free form of RF3 ognition of a stop signal in the ribosomal decoding site in in vitro termination assays (Goldstein & Caskey, by a protein release factor (RF), a form of molecular 1970). It was deduced that nucleotide-free RF3 stimu- mimicry of the tRNA that decodes sense codons dur- lated in vitro termination by increasing the affinity of ing translational elongation (Moffat & Tate, 1994; Ito RF1 and RF2 for the stop codon–ribosome complex, et al., 1996; Nakamura et al., 1996). This step is fol- and that the addition of GTP or GDP dissociated this lowed by hydrolysis of the terminal peptidyl-tRNA complex (Goldstein & Caskey, 1970). When the gene bond, so that the nascent polypeptide is released. In for RF3 was finally isolated, the amino acid sequence Escherichia coli, this process is mediated by RFs of two clearly indicated that RF3 is a member of the G protein types, the class I (or decoding factors), RF1 and RF2, superfamily, and has several regions of significant sim- and the class II (or stimulatory factor), RF3 (Pel et al., ilarity to EF-G (Grentzmann et al., 1994; Mikuni et al., 1996; Tate et al., 1996). 1994; Kawazu et al., 1995). E. coli RF3 was initially identified in crude extracts The fact that RF3 has a typical G domain was con- as a protein that stimulated the in vitro termination sistent with early studies indicating that guanine nu- activity of RF1 and RF2 (Capecchi & Klein, 1969; Mil- cleotides affected the function of nucleotide-free RF3. man et al., 1969). An important early finding in the The actual effects of these nucleotides, however, seemed characterization of RF3 was that the stimulatory activ- paradoxical in light of the usual effects of these nucle- otides on G protein function. The basic function of G proteins is to switch between two alternative confor- Reprint requests to: Warren P. Tate, Department of Biochemistry mational states, depending on whether GTP or GDP is and Centre for Gene Research, University of Otago, P.O. Box 56, Dunedin, New Zealand; e-mail: [email protected]. bound in the active site (Bourne et al., 1991). The switch *The first two authors contributed equally to the work. is activated by the GTP-hydrolysis activity of the 47 Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press 48 H.J. Pel et al. G-domain, and external effectors determine whether key, 1970). When RF2 is in excess, this effect is not ev- or not the hydrolysis reaction occurs. GTP and GDP ident (for example, compare Fig. 2A, lanes 5 and 6). are therefore expected to show opposite effects and the These results indicate that codon-dependent RF2 bind- observation that both nucleotides render RF3 inactive ing to the ribosome exhibits positive cooperativity with in termination assays in vitro has remained unexplained. nucleotide-free RF3. In this paper, we have examined the effect of gua- nine nucleotides on the binding of RF3 and RF2 to the Association of guanine nucleotide states ribosome to resolve the paradox of how these nucle- of RF3 with the ribosome otides can modulate the function of RF3. We show that RF3, like a classical G protein, switches between GTP What are the effects of guanine nucleotides on the (GDPCP)-bound “active” and GDP-bound “inactive” interaction of RF2 and RF3 with the ribosome and on states. As this work was being prepared for publica- the cooperativity of this interaction? Because GTP is tion, Freistroffer et al. (1997) reported, using ribosome rapidly hydrolyzed in the presence of RF3 and ribo- complexes of peptidyl-tRNA and synthetic mRNA, that somes (unpubl., Freistroffer et al., 1997), we chose the RF3 together with GTP stimulated decoding factor- nonhydrolysable GTP analogue, GDPCP, to fix the con- mediated peptide release. formation of RF3 in the GTP state. As illustrated in Figure 3A, the presence of GDPCP increased the bind- ing of RF3 to ribosomes four- to fivefold compared to RESULTS the nucleotide-free condition. GDP, on the other hand, consistently caused a reduction in the RF3 binding. Association of nucleotide-free The GTP analogue form of RF3 has a 10-fold higher RF3 with the ribosome affinity for the ribosome when compared to RF3{GDP. E. coli RF3 is structurally equivalent to the G domain This experiment was the first indication that RF3 be- and domains II and III of EF-G (see Fig. 1). The ab- haves similarly to the other translational G proteins in sence of the EF-G structural domains IV and V from that the protein is capable of switching between a high- RF3 suggests that it can bind to the ribosome simul- affinity GTP-induced conformation and a GDP-induced taneously with a class I RF, which we have previously low-affinity state. However, it adds to the puzzle of proposed binds to the A-site of the ribosome like tRNA why GTP inhibited the RF2-mediated release of fMet and may be equivalent to domains IV and V of EF-G from termination complexes when the nucleotide-free (Fig. 1) (Nakamura et al., 1996). The implication of this form was strongly stimulatory (Goldstein & Caskey, is that the decoding RFs (1 or 2) and RF3 have adja- 1970). cent, possibly interacting, binding sites on the ribo- Next we examined the ability of the guanine nucle- some. For this study, we have developed a simple direct otides to modulate the cooperativity of RF2 and RF3 assay to analyze the interaction of RF3 with the ribo- binding to the ribosome. The effects of GDP and the some. Radiolabeled RFs were prepared by metaboli- GTP analogue on stimulation of RF3 binding by RF2 cally labeling separate E. coli cultures using controlled are shown in Figure 3A (hatched bars). The decrease in overexpression of RFs in the presence of 35S-methionine. the binding of RF3 alone with GDP was partly restored Although the radiolabeled RF3 was significantly less in the presence of RF2. In contrast, the strong stimula- pure than the RF2 preparation, the partially purified tion of RF3 ribosomal binding by the GTP analogue 35S-RF3 fraction gave a single radiolabeled band asso- (Fig. 3A, four- to fivefold, compare lanes 1 and 5) was ciating with the ribosomes in the binding reactions greater than that given by RF2 without the nucleotide (Fig. 2A, lane 1). Because the early reports of RF3 func- (Fig. 3A, threefold, compare lanes 1 and 2), and the ad- tion (for example, Goldstein & Caskey, 1970) studied dition of RF2 in combination with the GTP analogue the nucleotide-free form of the protein, our initial stud- gave little further enhancement of RF3-ribosome bind- ies were performed in the absence of guanine nucleo- ing (Fig. 3A, compare lanes 5 and 6). This experiment tides. The binding of nucleotide-free RF3 to the ribosome also illustrates that the nucleotide-free form of RF3 has was only significantly enhanced by the presence of intermediate affinity between that of the RF3{GDPCP a fixed amount of RF2 when the stop codon, UGA, was and RF3{GDP. also present (Fig. 2A, lane 6 compared with lanes 1 In order to determine the effects of nucleotide-bound and 3). In the absence of RF2, RF3 binding to ribosomes RF3 on the binding of the class I RFs, we tested how was not significantly affected by UGA alone (lane 4). Ti- RF3 in the GTP- and GDP-bound states affected the tration of nucleotide-free RF3 with limiting concentra- sequestering of radiolabeled RF2 on the ribosome, as tions of RF2 results in a more than twofold increase shown in Figure 3B.
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