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

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 nucleotides; 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 nucleotides 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 guanine 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 binding 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. Nucleotide-free RF3 increased the in RF2 on the ribosome (Fig. 2B). This result explains binding of 35S-RF2 to ribosomes, as shown in Fig- why the early studies indicated a stimulatory effect of ure 2B, and this is reproduced in this second experi- nucleotide-free RF3 on the RF2-mediated release of ment. As expected, GDP, which we predict would fMet from a termination complex (see Goldstein & Cas- induce an “inactive” RF3 conformation, abolished the Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

Guanine nucleotide control of RF3 activity 49

FIGURE 1. Similarity between E. coli RF3 and EF-G. A: Schematic representation of sequence similarity between E. coli EF-G (GenBank accession no. X00415) and RF3 (D17724). Sequences were aligned using Bestfit (GCG package). Bars indicate positions of sequence identity and numbers II–V indicate structural domains of EF-G (Ævarsson et al., 1994). RF3 shows similarity to the EF-G domains G, G9, II, III, and part of domain IV. B: Resolution of the crystal structure of EF-G (Ævarsson et al., 1994; Cworkowski et al., 1994) and the phenyl-tRNA{EF-Tu{GDPNP complex (Nissen et al., 1995) revealed that EF-G domains IV and V mimic the aminoacyl-tRNA present in the EF-Tu complex. The figure displays the tertiary structure of Thermus thermophilus EF-G (Ævarsson et al., 1994) and yeast tRNAPhe. The tRNA is mimicked by EF-G domains III, IV, and V. RF1 and RF2 function as tRNA analogues and may mimic tRNA in the region that corresponds to EF-G domains IV and V (Moffat & Tate, 1994; Ito et al., 1996). positive effect of RF3 on RF2-ribosome binding. Con- RF3 inhibits in vitro termination under trary to our expectation, however, a similar effect was rate enhancing conditions also seen with GDPCP. The presence of this nucleotide even tends to dissociate RF2 from the ribosome, Overall, the above experiments suggest that RF3 bind- a result compatible with an earlier observation that ing to ribosomes is regulated by the GTP/GDP con- GDPCP can trigger the dissociation of radiolabeled formational switch similar to a typical G protein. termination codon from termination complexes (Gold- Therefore, we re-examined the effects of RF3-guanine stein & Caskey, 1970). Hence, the cooperativity observed nucleotide complexes on class I release factor-mediated in ribosome binding of RF2 and nucleotide-free RF3 was codon-dependent peptidyl-tRNA hydrolysis in vitro, not found in the presence of the GTP analogue. the assay used in the original studies. In this assay, Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

50 H.J. Pel et al.

FIGURE 3. Effects of guanine nucleotides on ribosome binding of RF3 and RF2. A: Effects of GDP and GDPCP on RF3-ribosome binding. 70S ribosomes were incubated with 10 pmol 35S-RF3 in the absence or presence of 20 pmol 35S-RF2, 1 mM GDP, or 1 mM GDPCP as indicated. Ribosome-associated 35S-RF3 was detected and quantitated as described in Figure 2. B: Effects of RF3, GDP, and GDPCP on the ribosome binding of RF2. Pure 35S-RF2 (10 pmol) was incubated with ribosomes alone or in combination with 50 pmol unlabeled RF3 in the absence or presence of 1 mM GDP and GDPCP 35 FIGURE 2. RF2 and RF3 bind the ribosome cooperatively. A: RF3- as indicated. Ribosome-associated S-RF2 was detected by ultra- ribosome binding is increased by codon-dependent RF2 binding. centrifugation followed by scintillation counting of the solubilized 70S ribosomes (50 pmol) was incubated with 20 pmol 35S-RF2, pellet. 10 pmol 35S-RF3 in the presence or absence of 10 mM UGA as indicated. Ribosome-associated 35S-RF2 and 35S-RF3 were detected by ultracentrifugation followed by SDS-PAGE of the solubilized ribo- hydrolysis of a model peptidyl-tRNA is strictly depen- somal pellet. A fluorograph and its densitometric quantitation are shown. B: RF2-ribosome binding is increased by RF3 binding. 70S dent on a class I release factor, and it requires either ribosomes (50 pmol) were incubated with 10 pmol 35S-RF2, 0–60 cognate stop codon trinucleotide or ethanol (5–10%) to pmol of 35S-RF3, in the presence of 10 mM UGA as indicated. promote termination complex formation between the Ribosome-associated RF2 and RF3 were detected and quantitated as described for A. release factor and the substrate complex (Scolnick & Caskey, 1969). Under these conditions, termination com- Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

Guanine nucleotide control of RF3 activity 51

ABC

FIGURE 4. Hydrolysis of f[3H]Met-tRNA in the absence or presence of ethanol and increasing amounts of termination codon. Termination assays contained 0.25 pmol RF1, ethanol as indicated, and contained (solid circles) or lacked (open circles) 20 pmol RF3. f[3H]Met released was extracted and quantitated as described in Materials and Methods. plex formation is the rate-limiting step, and nucleotide- like GDP in relieving the inhibition of RF3, consistent free RF3 has been shown to stimulate the release with a fast ribosome-mediated hydrolysis (Fig. 5, reaction by favoring codon-dependent complex forma- lanes 3 and 5). Nucleotide-free RF3 again shows an tion, a phenomenon that we can now explain by the effect that is intermediate to the effects of GDP and cooperative nature with which decoding RF and GDPCP (Fig. 5, compare lane 2 with lanes 3 and 4). nucleotide-free RF3 bind to the ribosome. Using this assay, GDP and GDPCP abolished the stimulatory ac- DISCUSSION tion of the nucleotide-free form of RF3, as previously reported (data not shown). The termination of protein synthesis is a unique and A termination complex stabilized by the presence of still poorly understood ribosomal reaction that in- ethanol could well be a better simulation of the in vivo volves subversion of the ribosome to catalyze a differ- situation where an mRNA presenting a stop signal at the A site is stably bound to the ribosome. We therefore investigated the effect of titrations of ethanol and termination codon on the termination efficiency in the presence of constant amounts of nucleotide-free RF3. Where the initial complex formation step is greatly enhanced by the inclusion of ethanol and UAG codon, the rate of class I RF-dependent peptidyl-tRNA hydrolysis reaction is also greatly enhanced (Fig. 4B,C). In- terestingly, the presence of ethanol reversed the effect of RF3. In the absence of ethanol (Fig. 4A), nucleotide- free RF3 stimulated the termination reaction (solid circles). When ethanol was added at 5%, RF3 did not further increase the rate of the reaction (Fig. 4B), and, at 10% ethanol, the reaction was significantly inhibited by RF3 (Fig. 4C). To examine this effect of RF3 further, we tested how guanine nucleotides modulated this inhibitory action of RF3. Significantly, GDP relieved the inhibition of RF3 (Fig. 5, compare lanes 2 and 4), consistent with the GDP-bound form having a low affinity for the ribosome. GDPCP, on the other hand, 3 significantly increases the inhibition of the in vitro ter- FIGURE 5. Hydrolysis of f[ H]Met-tRNA on stabilized termination complexes. Termination assays contained 10 mM UAG, 0.25 pmol mination reaction shown by the nucleotide-free form RF1, 20 pmol RF3 (black bars) or no RF3 (hatched bar), 10% (v/v) of RF3 (Fig. 5, compare lanes 2 and 4). GTP behaved ethanol, and 1 mM of the indicated guanine nucleotides. Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

52 H.J. Pel et al. ent reaction, hydrolysis rather than peptidyltransfer, pears as an inhibition when compared to the activity in response to a different decoding molecule, the RF of nucleotide-free RF3 because the latter has a stimu- protein rather than tRNA. Although it has been useful latory effect in the same assay. historically to draw parallels between termination and One of the most interesting findings of our study elongation, they required “leaps of faith.” With recent was the reversal of the enhancement by nucleotide- progress, we can now begin to address more realisti- free RF3 of fMet release upon stabilization of the ter- cally such analogies. The pertinent issues are whether mination complex by ethanol. The inhibitory effect of the class I RFs are molecular mimics of tRNA, and RF3 observed with the stabilized complex is in good whether class II RFs, such as RF3, function analo- agreement with the inhibitory effect that Freistroffer gously to the elongation factors EF-Tu and EF-G. et al. (1997) observed for nucleotide-free RF3 in their The experiments presented in this paper provide ev- termination assay. Goldstein and Caskey (1970) ob- idence that RF3 is not only structurally, but also mech- served the same effect when they stabilized the termi- anistically similar to EF-G and EF-Tu. Most importantly, nation complex by using very high termination codon ϩ ϩ this is the first direct demonstration of RF3 binding to concentrations in the presence of NH4 instead of K ribosomes in its guanine nucleotide forms and the bind- ions. ing seems to be regulated by conformational switching Freistroffer et al. (1997) recently presented compel- between a high-affinity GTP and a low-affinity GDP ling evidence that RF3 did not accelerate the associa- state. These data are more in line with the accepted tion of RF1 and RF2 with the ribosome or the rate of mechanism of translational G proteins than the previ- peptidyl-tRNA hydrolysis, and therefore concluded that ous experiments, which presented the paradox that RF3 must accelerate the dissociation of the decoding RF3 activity was only routinely detectable in the ab- factors. Based on their data and this conclusion, they sence of nucleotide. Freistroffer et al. (1997) have also have proposed a provocative and valuable model for shown that RF3 in concert with GTP acts as a classical RF3 action in which the protein binds after peptidyl- G protein in stimulating the release of a peptide in a tRNA hydrolysis. Our observation that the GDPCP an- series of elegant experiments with a short designed alogue inhibited peptide release under conditions where mRNA. the decoding factor was limiting is consistent with this The data obtained in our study indicate that RF3{GTP finding, although the model presented by Freistroffer (as the analogue, GDPCP) and class I RFs can bind et al. (1997) invokes the GDP state to bind stably to the alone or simultaneously to the ribosome. The cooper- ribosome, whereas we show in this study that RF3{GDP ative binding activity of nucleotide-free RF3 might be has low affinity for the ribosome. due to this form of the protein being in a flexible How does the mechanism of RF3 compare with that conformational intermediate between the tightly ribo- of EF-Tu, one of the G proteins involved in transla- some-bound GTP conformation, which can bind non- tional elongation? If RF3 mimicked EF-Tu, then it would cooperatively, and the dissociated GDP state (see, for bind to the posttranslocational ribosome in a ternary example, Figs. 3A and 5). Now that we know that RF3 complex with GTP and a decoding release factor (Nier- is a true member of the family of translational G pro- haus 1993, 1996). However, our results indicate that, teins, it seems very unlikely that RF3 is ever present in unlike EF-Tu, RF3 does not form an extraribosomal the cell in a nucleotide-free state, except perhaps in a ternary complex with GTP and a decoding molecule in transient state. The cooperative binding and rate- order to deliver that decoding molecule to the ribo- enhancing activity of nucleotide-free RF3 suggests that some. Rather, we have shown that the decoding RF it mimics a transition state of RF3 in which the protein and RF3 bind to the ribosome independently of each interacts with the decoding release factor while it en- other when a GTP analogue is present. There is as yet hances the efficiency of the termination reaction. Thus, no strong evidence that the two classes of prokaryotic it appears that, although the activity of nucleotide-free release factors form a complex independent of the RF3 may not reflect a true physiological state, it is a ribosome despite our rigorous efforts to detect such serendipitous one because it allowed the discovery of complexes (W.P. Tate & Y. Nakamura unpubl.). In eu- RF3 in the late 1960s. karyotes, however, the decoding release factor eRF1 How can the enhanced binding of the GDPCP form does form a complex off the ribosome with the G pro- of RF3 over the nucleotide-free form of the protein be tein eRF3, suggesting that the two factors bind the reconciled with the earlier studies of an apparent lack ribosome in a complex with GTP, thus mimicking the of stimulation of the in vitro termination reaction by eEF1A (EF-1a) ternary complex (Stansfield et al., 1995). RF3{GTP? An important clue to this disparity might On the ribosome, eRF3 functions in a ternary complex come from the observation of Freistroffer et al. (1997) with decoding factor and GTP (Frolova et al., 1996). that GTP is quickly hydrolyzed in the presence of RF3 How does the mechanism of RF3 compare with that of and naked ribosomes or termination complexes. Our EF-G, the other G protein involved in translational data clearly indicate that the resulting GDP form of elongation? During the elongation process, EF-G binds RF3 lacks stimulatory activity. This lack of activity ap- to the pretranslocational ribosome after peptide trans- Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

Guanine nucleotide control of RF3 activity 53 fer has occurred (Nierhaus 1993, 1996). If hydrolysis of gels. The gels were Coomassie-stained, then soaked in En- peptidyl-tRNA during termination resembles peptide hance (Amersham), dried, and exposed to Cronex film. In- transfer during elongation, then an EF-G like role for tensities of bands on the fluorographs were determined by RF3 predicts that the protein binds after peptidyl- scanning densitometry and image analysis using NIH-Image tRNA hydrolysis, a prediction that is consistent with software. the data we present in this paper. In summary, the currently available data seem to indicate that, whereas Termination assay RF3 may resemble both EF-Tu and EF-G in some functional aspects, it appears to be a unique translational G In vitro termination was assayed as described previously protein in many others. The finer points of its mech- (Mikuni et al., 1994). The substrate complex containing 3 anism should emerge rapidly now that recombinant f[ H]Met-tRNA in the ribosomal P-site was prepared by in- protein is available and better in vitro systems of ter- cubating 50 pmol 70S ribosomes, 2.5 nmol AUG, and 25 pmol f[3H]Met-tRNA in a 50-mL reaction containing 20 mM mination are being developed. Tris-HCl, pH 7.5, 0.15 M NH4Cl, and 10 mM MgCl2 at 30 °C for 30 min. To detect termination, 5 mL of substrate was MATERIALS AND METHODS added to a 50-mL reaction containing indicated amounts of release factors, trinucleotide stop codon, and ethanol, and Production of 35S-labeled RF proteins incubated for 30 min at 20 °C. To terminate the reaction, 200 mL of 0.1 M HCl was added, free f[3H]Met was extracted E. coli strain MP347 carrying RF3 expression plasmid pTOSOP into 1 mL of ethyl acetate, and the radioactivity was deter- was grown at 32 °C in M9 minimal medium supplemented mined. with chloramphenicol (10 mg/mL), 2 mg/mL biotin, and 40 mg/mL each of 18 amino acids (no methionine and cys- ACKNOWLEDGMENTS teine). At mid-logarithmic phase, RF3 expression was induced by shifting to 42 °C. Cold methionine (1 mg/mL) and We thank Dr. Richard Buckingham and Dr. Guido Grentz- 35 0.5 mCi [ S]-methionine were added at the time of heat mann for making data available prior to publication, and shock. After4hgrowth at elevated temperature, cells were Yoichi Kawazu and Sally Mannering for critical reading of harvested, washed, and suspended in lysis buffer (50 mM the manuscript. The work was supported by a Human Fron- Tris-Cl, pH 8.0, 100 mM NaCl, 1 mM DTT, 2 mM EDTA, tiers Science Program grant to Yoshi Nakamura and Warren 2 mM phenylmethylsulfonic acid) containing lysozyme Tate, who is an International Scholar of the Howard Hughes (1 mg/mL). After 20 min on ice, sodium deoxycholate (0.04%) Medical Institute. and DNAse I (2 mg/mL) were added and incubation was continued for 10 min. The lysate was clarified by centrifu- Manuscript accepted without revision October 22, 1997 gation at 12,000 ϫ g for 10 min, followed by 90 min at 60,000 rpm in the 75Ti rotor. The supernatant was fractionated by stepwise ammonium sulfate precipitation. The material pre- REFERENCES cipitating between 55% and 80% saturation was dissolved in a minimal volume of 50 mM Tris-HCl, pH 7.8, 0.1 M KCl, Ævarsson A, Brazhnikov E, Garber M, Zheltonosova J, Chirgadze Y, Al-Karadaghi S, Svensson LA, Liljas A. 1994. Three-dimensional 10 mM MgCl2, and 1 mM dithiothreitol and desalted by gel structure of the ribosomal translocase: Elongation factor G from filtration on a column of Sephadex G-25 equilibrated in the Thermus thermophilus. EMBO J 13:3669–3677. same buffer. Bourne HR, Sanders DA, McCormick F. 1991. The GTPase super- 35S-RF2 was prepared from E. coli JM105 carrying the RF2 family: Conserved structure and molecular mechanism. Nature 349:117–127. expression plasmid pRF2* (Donly et al., 1990) as previously Capecchi MR, Klein HA. 1969. Characterization of three proteins described (Tate et al., 1990) and was greater than 95% pure as involved in polypeptide chain termination. Cold Spring Harbor assessed by SDS-PAGE and fluorography. Symp Quant Biol 34:469–477. Cworkowski J, Wang J, Steitz TA, Moore PB. 1994. The crystal structure of elongation factor G complexed with GDP, at 27 Å reso- Ribosome binding assay lution. EMBO J 13:3661–3668. Donly BC, Edgar CD, Adamski FM, Tate WP. 1990. Frameshift auto- 35S-labeled RF2 and RF3 proteins (10–20 pmol) were incu- regulation in the gene for Escherichia coli release factor 2 —Partly functional mutants result in frameshift enhancement. Nucleic Acids bated with 50 pmol 70S ribosomes in a 200-mL reaction con- Res 18:6517–6522. taining 4% (v/v) ethanol, 0.1 mg/mL bovine serum albumin, Freistroffer DV, Pavlov MY, MacDougall J, Buckingham RH, Ehren- 50 mM Tris-HCl, pH 7.2, 75 mM KCl, and 30 mM MgCl2 in berg M. 1997. Release factor RF3 in E. coli accelerates the disso- siliconized Beckman airfuge tubes. Samples were incubated ciation of release factors RF1 and RF2 from the ribosome in a at 4 °C for 20 min, then centrifuged for 15 min at 95,000 rpm GTP-dependent manner. EMBO J 13:4126–4133. Frolova L, Le Goff X, Zhouravleva G, Davydova E, Philippe M, in a Beckman Airfuge at 4 °C. Free and ribosome-bound RF2 Kisselev L. 1996. Eukaryotic polypeptide chain release factor eRF3 were measured by scintillation counting of the supernatant is an eRF1- and ribosome-dependent guanosine triphosphatase. and pellet, solubilized in 5% SDS. To determine ribosome- RNA 2:334–341. bound RF3, supernatants were carefully and thoroughly re- Goldstein JL, Caskey CT. 1970. Peptide chain termination: Effect of protein S on ribosomal binding of release factors. Proc Natl Acad moved and pellets dissolved in 20 mL of gel loading dye Sci USA 67:537–543. (6 M urea, 1% SDS, 20 mM Tris-HCl, pH 6.7) heated to 65 °C. Grentzmann G, Brechemier-Baey D, Heurgué V, Mora L, Bucking- Samples were electrophoresed on 10% SDS-polyacrylamide ham RH. 1994. Localisation and characterisation of the gene en- Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

54 H.J. Pel et al.

coding release factor RF3 in Escherichia coli. Proc Natl Acad Sci Nierhaus KH. 1996. An elongation factor turn-on. Nature 379:491– USA 91:5848–5852. 492. Ito K, Ebihara K, Uno M, Nakamura Y. 1996. Conserved motifs in Nissen P, Kjeldgaard M, Thirup S, Polekhina G, Reshetnikova L, prokaryotic and eukaryotic polypeptide release factors: tRNA- Clark BFC, Nyborg J. 1995. Crystal structure of the ternary protein mimicry hypothesis. Proc Natl Acad Sci USA 93:5443– complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 5448. 270:1464–1472. Kawazu Y, Ito K, Matsumura K, Nakamura Y. 1995. Comparative Pel HJ, Rozenfeld S, Bolotin-Fukuhara M. 1996. The nuclear Kluyvero- characterization of release factor RF3 genes of Escherichia coli, myces lactis MRF1 gene encodes a mitochondrial peptide chain Salmonella typhimurium, and Dichelobacter nodosus. J Bacteriol release factor that is important for cell viability. Curr Genet 30:19– 177:5547–5553. 28. Mikuni O, Ito K, Moffat J, Matsumura K, McCaughan K, Nobukuni Scolnick EM, Caskey CT. 1969. Peptide chain termination, V. The T, Tate W, Nakamura Y. 1994. Identification of the prfC gene, role of release factors in mRNA terminator codon recognition. which encodes peptide chain release factor 3 of Escherichia coli. Proc Natl Acad Sci USA 64:1235–1239. Proc Natl Acad Sci USA 91:5798–5802. Stansfield I, Jones K, Kushnirov VK, Dagkesamanskaya AR, Poznya- Milman G, Goldstein J, Scolnick E, Caskey CT. 1969. Peptide chain kovski AI, Paushkin SV, Nierras CR, Cox BS, Ter-Avanesyan MD, termination, III. Stimulation of in vitro termination. Proc Natl Tuite MF. 1995. The products of the SUP45 (eRF1) and SUP35 Acad Sci USA 63:183–190. genes interact to mediate translation termination in Saccharomy- Moffat JG, Tate WP. 1994. A single proteolytic cleavage in release ces cerevisiae. EMBO J 14:4365–4373. factor 2 stabilizes ribosome binding and abolishes peptidyl- Tate WP, Dalphin ME, Pel HJ, Mannering SA. 1996. The stop signal tRNA hydrolysis activity. J Biol Chem 269:18899–18903. controls the efficiency of release factor-mediated translational Nakamura Y, Ito K, Isaksson LA. 1996. Emerging understanding of termination. Genetic Eng 18:157–182. translation termination. Cell 87:147–150. Tate WP, Greuer B, Brimacombe R. 1990. Codon recognition in poly- Nierhaus KH. 1993. Solution of the ribosome riddle: How the ribo- peptide chain termination: Site directed crosslinking of termina- some selects the correct aminoacyl-tRNA out of 41 contestants. tion codon to Escherichia coli release factor 2. Nucleic Acids Res Mol Microbiol 9:661–669. 18:6537–6544. Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides.

H J Pel, J G Moffat, K Ito, et al.

RNA 1998 4: 47-54

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

To subscribe to RNA go to: http://rnajournal.cshlp.org/subscriptions