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Release of Ribosome-bound Ribosome Recycling Factor by Elongation Factor G

2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês

10.1074/jbc.m304834200

1083-351X

Michael Kiel, V. Samuel Raj, Hideko Kaji, Akira Kaji,

Toxin Mechanisms and Immunotoxins

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site. Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site. Protein synthesis occurs on ribosomes in three basic steps of initiation (1Kozak M. Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1116) Google Scholar, 2Gualerzi C.O. Brandi L. Caserta E. La Teana A. Spurio R. Tomsic J. Pon C.L. Garret R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. American Society for Microbiology Press, Washington, D. C.2000: 477-494Google Scholar), elongation (3Nierhaus K.H. Spahn C. Burkhardt N. Dabrowski M. Diedrich G. Einfeldt E. Kamp D. Marquez V. Patzke S. Schafer M.A. Stelzl U. Blaha G. Willumeit R. Stuhrmann H.B. Garret R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. American Society for Microbiology Press, Washington, D. C.2000: 319-335Google Scholar, 4Rodnina M.V. Pape T. Savelsbergh A. Mohr D. Matassova N.B. Wintermeyer W. Garret R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. American Society for Microbiology Press, Washington, D. C.2000: 301-317Google Scholar, 5Wintermeyer W. Savelsbergh A. Semenkov Y.P. Katunin V.I. Rodnina M.V. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 449-458Crossref PubMed Scopus (25) Google Scholar), and termination (6Wilson D.N. Dalphin M.E. Pel H.J. Major L.L. Mansell J.B. Tate W.P. Garret R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. American Society for Microbiology Press, Washington, D. C.2000: 495-508Google Scholar). Both deacylated tRNA and mRNA remain bound to the ribosome after the termination step (7Hirashima A. Kaji A. Biochem. Biophys. Res. Commun. 1970; 41: 877-883Crossref PubMed Scopus (39) Google Scholar, 8Ogawa K. Kaji A. Eur. J. Biochem. 1975; 58: 411-419Crossref PubMed Scopus (29) Google Scholar, 9Janosi L. Mottagui-Tabar S. Isaksson L.A. Sekine Y. Ohtsubo E. Zhang S. Goon S. Nelken S. Shuda M. Kaji A. EMBO J. 1998; 17: 1141-1151Crossref PubMed Scopus (116) Google Scholar). In order for the ribosome to enter a new round of translation, it must be "recycled," a process achieved by the release of both tRNA and mRNA. This "fourth step" of protein synthesis is catalyzed by the concerted action of elongation factor G (EF-G) 1The abbreviations used are: EF-Gelongation factor GRRFribosome recycling factorDTTdithiothreitolATPγSadenosine 5′-O-(thiotriphosphate)GMP-PCPguanylyl β,γ-methylenediphosphonate.1The abbreviations used are: EF-Gelongation factor GRRFribosome recycling factorDTTdithiothreitolATPγSadenosine 5′-O-(thiotriphosphate)GMP-PCPguanylyl β,γ-methylenediphosphonate. and ribosome recycling factor (RRF) (reviewed in Refs. 10Janosi L. Hara H. Zhang S. Kaji A. Adv. Biophys. 1996; 32: 121-201Crossref PubMed Scopus (104) Google Scholar and 11Kaji A. Kiel M.C. Hirokawa G. Muto A. Inokuchi Y. Kaji H. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 515-529Crossref PubMed Scopus (37) Google Scholar). The simultaneous presence of EF-G and RRF is required (12Hirashima A. Kaji A. J. Biol. Chem. 1973; 248: 7580-7587Abstract Full Text PDF PubMed Google Scholar), and optimal activity occurs when they are present at a 1:1 ratio (13Hirashima A. Kaji A. Biochemistry. 1972; 11: 4037-4044Crossref PubMed Scopus (45) Google Scholar). elongation factor G ribosome recycling factor dithiothreitol adenosine 5′-O-(thiotriphosphate) guanylyl β,γ-methylenediphosphonate. elongation factor G ribosome recycling factor dithiothreitol adenosine 5′-O-(thiotriphosphate) guanylyl β,γ-methylenediphosphonate. RRF is an essential protein for cell growth (14Janosi L. Shimizu I. Kaji A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4249-4253Crossref PubMed Scopus (103) Google Scholar). It is a nearly perfect structural (15Selmer M. Al-Karadaghi S. Hirokawa G. Kaji A. Liljas A. Science. 1999; 286: 2349-2352Crossref PubMed Scopus (152) Google Scholar) and functional (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar) tRNA mimic. However, RRF lies at the ribosomal subunit interface, across the 50 S subunit, in a position that is nearly orthogonal to the A- and P-site bound tRNA (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 18Lancaster L. Kiel M.C. Kaji A. Noller H.F. Cell. 2002; 111: 129-140Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These data rule out a straightforward functional mimicry of tRNA by RRF but do not rule out a possible movement of RRF by EF-G (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). In this paper, we describe how EF-G affects the binding of RRF to ribosomes. RRF readily forms complexes with 70 S ribosomes, 50 S ribosomal subunits, and polysomes (also see Refs. 16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar and 17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). We demonstrate that, as part of the RRF-dependent post-termination complex disassembly reaction, EF-G releases RRF from ribosomal complexes, and this release activity of EF-G is due to the translocation activity of EF-G. Buffers—The buffers used were as follows: buffer A, 20 mm Tris-HCl (pH 7.5), 100 mm NH4Cl, 10 mm Mg(OAc)2, 3 mm DTT; buffer B, 20 mm Tris-HCl (pH 7.5), 10 mm Mg(OAc)2, 500 mm NH4Cl, 2 mm DTT; buffer C, 20 mm Tris-HCl (pH 7.5), 50 mm NH4Cl, 10 mm Mg(OAc)2, 2 mm DTT; buffer D, 10 mm Tris-HCl (pH 7.5), 10 mm MgSO4, 50 mm NH4Cl, 0.5 mm DTT; buffer E, 20 mm Tris-HCl (pH 7.5), 100 mm KCl, 1 mm DTT; buffer F, 5 mm potassium phosphate buffer (pH 7), 1 mm DTT; buffer G, 50 mm Tris-HCl (pH 7.5), 25 mm KCl, 10 mm Mg(OAc)2; buffer H, 14 mm Tris-HCl (pH 7.4), 12 mm Mg(OAc)2, 66 mm NH4Cl, 0.3 mm DTT; buffer I, 50 mm Tris-HCl (pH 8.0), 100 mm KCl, 7 mm Mg(OAc)2, 8 m urea; buffer J, 50 mm Tris-HCl (pH 7.5), 100 mm KCl, 7 mm Mg(OAc)2, 300 mm imidazole. Preparation of 70 S Ribosomes—17 g of Escherichia coli MRE 600 cells (purchased from the University of Alabama Fermentation Facility) were thawed, suspended in 35 ml of buffer A, and passed once through a French press at ∼12,000 p.s.i. The cell suspension was centrifuged twice at 30,000 × g for 15 min, discarding the pellets. The supernatant was layered onto four 15-ml sucrose solutions (20 mm Tris-HCl (pH 7.5), 10 mm Mg(OAc)2, 500 mm NH4Cl, 2 mm DTT, 1.1 m sucrose) and centrifuged at 30,000 rpm in a Ti70 rotor for 22 h. The crude ribosome pellets were resuspended in 45 ml of buffer B (no sucrose), cleared by a 15-min centrifugation step at 30,000 × g, and pelleted at 35,000 rpm in a Ti70 rotor for 2 h. The clearing and pelleting step was repeated once more. The washed ribosome pellet was resuspended in storage buffer (buffer A except 50 mm NH4Cl) and stored at –80 °C. Analysis of the 70 S ribosomes on 10–30% sucrose gradients (8 mm Mg2+) showed no significant subunit dissociation of the 70 S ribosomes. Purification of Native RRF—Native RRF was purified from an over-producing strain, DH5α(pRR2) (19Shimizu I. Kaji A. J. Bacteriol. 1991; 173: 5181-5187Crossref PubMed Google Scholar), as we have previously described (13Hirashima A. Kaji A. Biochemistry. 1972; 11: 4037-4044Crossref PubMed Scopus (45) Google Scholar). The purified RRF was dialyzed against buffer D and stored at –80 °C. Purification of Native EF-G—Native EF-G was purified from an overproducing strain, JM83(pECEG) based on published methods (20Kaziro Y. Inoue N. J. Biochem. (Tokyo). 1968; 64: 423-425Crossref PubMed Scopus (19) Google Scholar, 21Hou Y. Yaskowiak E.S. March P.E. J. Bacteriol. 1994; 176: 7038-7044Crossref PubMed Google Scholar). Four grams of cells were lysed in buffer C by passing through a French press at 12,000 p.s.i. The cell suspension was centrifuged once at 30,000 × g for 20 min (pellet discarded) and once at 40,000 rpm in a Ti70 rotor for 2 h. The supernatant was precipitated with 70% saturated (NH4)2SO4 and resuspended in 8 ml of buffer E. The suspension was passed through a Sephadex G100 column equilibrated with buffer E. Fractions containing EF-G were determined by Coomassie-stained SDS gels, pooled, and applied to a 22-ml DEAE-Sepharose column equilibrated in buffer E. EF-G was eluted with a 0–0.6 m KCl gradient in buffer E. Fractions containing EF-G were pooled, dialyzed against buffer F, and applied to a 10-ml hydroxylapatite column equilibrated in buffer F. Purified EF-G was eluted with a 5–500 mm phosphate gradient (pH 7), dialyzed against buffer D, and stored at –80 °C. Purification of Mutant EF-G—BL21(DE3) E. coli cells harboring plasmids expressing His-tagged mutant EF-G, lacking single domains (either domain 1, 4, or 5) or having a single point mutation (H583K) (22Borowski C. Rodnina M. Wintermeyer W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4202-4206Crossref PubMed Scopus (43) Google Scholar, 23Rodnina M.V. Savelsbergh A. Katunin V.I. Wintermeyer W. Nature. 1997; 385: 37-41Crossref PubMed Scopus (383) Google Scholar, 24Savelsbergh A. Matassova N.B. Rodnina M.V. Wintermeyer W. J. Mol. Biol. 2000; 300: 951-961Crossref PubMed Scopus (94) Google Scholar), were plated on LB medium with the appropriate antibiotic (either 125 μg/ml ampicillin or 30 μg/ml kanamycin). Individual colonies were then grown in LB liquid at 37 °C until an A600 ∼ 0.6 was reached. Isopropyl-1-thio-β-d-galactopyranoside was added at a final concentration of 1 mm, and the cells were grown an additional 3.5 h. The cells were harvested and lysed in buffer I. The His-tagged EF-G was bound to Ni2+-nitrilotriacetic acid beads (Qiagen) and washed in buffer I. His-tagged EF-G was refolded by slowly replacing buffer I with buffer I containing 1 m urea. The refolded EF-G was eluted with buffer J, dialyzed against buffer D, and stored at –80 °C. Preparation of RRF-Ribosome Complexes—70 S ribosomes (0.25–0.5 μm) and RRF (3.75–5 μm) were incubated together for 10 min at room temperature in 40 μl of buffer G. Since the Kd value of RRF to the vacant ribosome was previously measured as ∼0.5 μm (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), under these conditions ribosomes are saturated with RRF and 1:1 complexes are formed. Complexes were separated from free RRF by Microcon-100 (Millipore) ultrafiltration, and isolated complexes were routinely measured for ribosome concentration (1 A260 unit = 23 pmol) and the amount of RRF bound (quantitative Western blot), as we have previously described (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Freshly made complexes were used right away (within 5 min of preparation). Preparation of 35S-Labeled tRNAPhe—tRNAPhe (7 nmol; Sigma) was incubated with 150 μCi of [35S]ATPγS (1000 Ci/mmol) and 20 units of T4 polynucleotide kinase (Invitrogen) in 50 μl of reaction buffer (supplied with the kinase by Invitrogen). The reaction mixture was incubated for 20 min at 37 °C, followed by a heat-inactivating step at 65 °C for 10 min. The tRNAPhe was precipitated with 2 volumes of ethanol at –20 °C and pelleted by centrifugation at 15,000 × g for 15 min. The pellet was rinsed twice with 500 μl of 70% ethanol, air-dried, and resuspended in 200 μl of buffer G. Preparation of tRNA-Ribosome Complexes—70 S ribosomes (0.25 μm) and 35S-labeled tRNAPhe (1.25 μm, ∼13,500 cpm/pmol) were incubated together for 10 min at room temperature in 40 μl of buffer G with or without one A260 unit of poly(U). During the process of making the complexes of tRNA and ribosomes, no EF-G or RRF were added. Complexes were separated from free tRNA by Microcon-100 ultrafiltration as previously described (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Release of RRF or tRNA by EF-G—EF-G was added to 40 μl of freshly prepared RRF-ribosome or tRNA-ribosome complexes in buffer G. Release was allowed to occur for 10 min at 35 °C or room temperature. Released RRF or released tRNA was separated from complexes on Microcon-100 as described for the formation of the complexes (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The amount of RRF still bound to ribosomes was measured by quantitative Western blot analysis of the complexes using anti-RRF antibody as described (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The amount of tRNA bound to ribosomes was measured by filtering through nitrocellulose filters and measuring 35S counts on the filter. Binding of RRF to 70 S Ribosomes in the Presence of EF-G—For studying the effects of the presence of EF-G on the binding of RRF to ribosomes, EF-G (15 or 120 pmol) was mixed with various concentrations of RRF, 10 pmol of 70 S ribosomes, and 1 mm GTP in 40 μl of buffer G. After the reaction, free EF-G and free RRF were removed from bound EF-G and RRF by Nano-Sep 300K ultrafiltration (Pall Life Sciences). The EF-G-ribosome complexes are not stable enough to withstand high speed centrifugation (about 80% is dissociated (25Cameron D.M. Thompson J. March P.E. Dahlberg A.E. J. Mol. Biol. 2002; 319: 27-35Crossref PubMed Scopus (83) Google Scholar)), but we found that they are stable enough to withstand isolation by Nano-Sep 300K ultrafiltration (details of the interaction between EF-G and ribosomes studied with this technique will be published elsewhere). The amounts of ribosome-bound EF-G and ribosome-bound RRF were determined by quantitative Western blot using anti-EF-G or anti-RRF antibody. In experiments where the binding of RRF to preformed complexes of ribosome, EF-G, and thiostrepton were studied, the preformed complexes were prepared by incubating 140 pmol of 70 S ribosomes, 300 pmol of EF-G, 1 mm GTP, and 40 μm thiostrepton in 40 μl of buffer G for 10 min. In the experiments where the binding of RRF to preformed complexes of ribosome, EF-G, and fusidic acid were studied, preformed complexes were prepared by incubating 100 pmol of ribosomes, 250 pmol of EF-G, 1 mm GTP, and 1 mm fusidic acid in 40 μl of buffer G for 10 min. In both cases, free EF-G was removed, bound EF-G was quantified, and the complexes were reacted with RRF as described above. RRF Assay with Model Post-termination Complexes—Polysome preparation and RRF assays were essentially as previously described (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). The RRF assay reaction mixture contained 2.2 A260 units of polysome per ml, 0.18 μm RRF, 0.55 μm EF-G, and 0.36 mm GTP (or other nucleotide) in buffer H. RRF binding to polysome and release was measured as described above for 70 S ribosomes except that a single Microcon-100 centrifugation at 3000 × g was used. No RRF was detected in the absence of polysome. tRNA release from the polysome was measured by amino-acylation of nitrocellulose (0.45 μm) filtrate as described (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). EF-G Releases Ribosome-bound RRF—Complexes of 70 S ribosomes and RRF were formed by incubating RRF with well washed, vacant ribosomes (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). RRF-ribosome complexes thus formed were isolated, and EF-G and GTP were added at various concentrations. As shown in Fig. 1A, EF-G releases RRF from ribosomes in a dose-dependent manner. Under these conditions, EF-G optimally works at an approximate 1:1 ratio with the complexes. This is consistent with earlier studies that had shown that the optimal rate of ribosome recycling occurs when EF-G and RRF are present at a 1:1 ratio (13Hirashima A. Kaji A. Biochemistry. 1972; 11: 4037-4044Crossref PubMed Scopus (45) Google Scholar), and it demonstrates that EF-G works stoichiometrically with RRF. One may wonder if EF-G actually releases RRF from the ribosomes or simply affects its rebinding in a rapid equilibrium situation. It should be noted that the Kd value of RRF to the vacant ribosome is 0.2–0.5 μm, and therefore only a fraction (∼30%) of the released RRF (∼0.2 μm maximum released) should rebind. In this experiment, however, one would expect that no rebinding would take place, because the presence of EF-G would constantly remove the rebound RRF. Since the Kd value of EF-G to the vacant ribosome is ∼0.04 μm, one would expect nearly 100% of ribosomes should have bound EF-G under the experimental conditions. It is possible that very rapid release and binding can take place, but such rapid action cannot be detected with the present technique used. However, in separate experiments with fluorescent RRF and fast kinetic analysis, we did not detect such a rapid action of release and rebinding. As indicated below, about 20–25% of the ribosomes used in this experiment are inactive with respect to EF-G binding. On the other hand, ∼100% of the ribosomes are able to bind RRF (one RRF per ribosome). Hence, about 25% of bound RRF remains on the ribosomes even in the presence of excess EF-G. In our previous studies, we showed that higher concentrations of NH4Cl inhibit the initial binding of RRF (e.g. 150 mm NH4Cl inhibits ∼70% of the initial binding of RRF as compared with 30 mm monovalent ions (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar)). It was therefore possible that RRF may be released at physiological monovalent concentrations (150 mm) without EF-G. As shown in Fig. 1B, higher NH4Cl concentrations could not substitute for EF-G activity, indicating that EF-G is required for the release of RRF from ribosomes under physiological monovalent ion conditions. EF-G Does Not Release tRNA Bound to Nonprogrammed Ribosomes—RRF is a nearly perfect structural mimic of tRNA (15Selmer M. Al-Karadaghi S. Hirokawa G. Kaji A. Liljas A. Science. 1999; 286: 2349-2352Crossref PubMed Scopus (152) Google Scholar). The effect of RRF on tRNA bound to nonprogrammed ribosomes, poly(U)-programmed ribosomes and polysomes, and vice versa has already been examined (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). We previously proposed that P/E site tRNA would be released by RRF and EF-G (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Furthermore, we showed that tRNA bound to ribosomes is released by EF-G only if the A-site is occupied (26Ishitsuka H. Kuriki Y. Kaji A. J. Biol. Chem. 1970; 245: 3346-3351Abstract Full Text PDF PubMed Google Scholar). Although the way RRF binds to ribosomes is quite different from that of tRNA, it would be of interest to examine whether tRNA is released from ribosomes by EF-G under similar conditions to the release of RRF. In the experiment described in Fig. 1C, complexes of ribosomes and tRNA were formed under conditions where tRNA binds to the P- and/or E-sites but not the A-site (26Ishitsuka H. Kuriki Y. Kaji A. J. Biol. Chem. 1970; 245: 3346-3351Abstract Full Text PDF PubMed Google Scholar, 27Igarashi K. Kaji A. Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 1971-1976Crossref PubMed Scopus (22) Google Scholar, 28Kirillov S.V. Makarov E.M. Semenkov Y.P. FEBS Lett. 1983; 157: 91-94Crossref PubMed Scopus (85) Google Scholar, 29Lill R. Robertson J.M. Wintermeyer W. Biochemistry. 1984; 23: 6710-6717Crossref PubMed Scopus (77) Google Scholar, 30Lill R. Robertson J.M. Wintermeyer W. Biochemistry. 1986; 25: 3245-3255Crossref PubMed Scopus (103) Google Scholar). It is clear from Fig. 1C that EF-G alone does not release the deacylated tRNA from these complexes as it does RRF. It is also noted that the presence or absence of poly(U) did not alter this situation. This is consistent with our earlier observation that the A-site must be occupied for the release of tRNA from the P- and/or E-site of nonprogrammed or homopolymer-programmed ribosomes by EF-G (26Ishitsuka H. Kuriki Y. Kaji A. J. Biol. Chem. 1970; 245: 3346-3351Abstract Full Text PDF PubMed Google Scholar), although the situation is different with natural mRNA (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). This experiment was performed to demonstrate that bound tRNA behaves differently from bound RRF upon the addition of EF-G and GTP. The effect of RRF and EF-G together on bound tRNA has already been studied in detail (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). Release of RRF Is Not Dependent on GTP Hydrolysis—The activity of RRF is routinely checked by a model post-termination complex disassembly assay (31Hirashima A. Kaji A. J. Mol. Biol. 1972; 65: 43-58Crossref PubMed Scopus (88) Google Scholar). In this assay, polysomes are treated with puromycin so that each ribosome acts as if it has reached a termination codon. The addition of RRF, EF-G, and GTP then converts the polysomes to monosomes by releasing both tRNA and mRNA. The hydrolysis of GTP is essential for the release of mRNA but not for the release of tRNA from model post-termination complexes (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). We therefore tested whether or not the hydrolysis of GTP by EF-G was also essential for the release of RRF from ribosomes. As shown in Table I, nonhydrolyzable GTP analogues were effective, indicating that GTP hydrolysis is not required for the release of RRF by EF-G and GTP. As a matter of fact, nonhydrolyzable GTP analogue gave the maximum release of RRF. GMP-PCP freezes EF-G on the ribosome (32Kuriki Y. Inoue N. Kaziro Y. Biochim. Biophys. Acta. 1970; 224: 487-497Crossref PubMed Scopus (45) Google Scholar), and this is the most likely reason why it gave the best release. The observation that the release of RRF takes place in the presence of GTP analogue has been confirmed by studies using fluorescent-labeled RRF. 2H. S. Seo, M. C. Kiel, V. S. Raj, A. Kaji, and B. S. Cooperman, manuscript in preparation.Table IThe release of RRF from ribosomes by EF-G is stimulated by guanine nucleotide, but GTP hydrolysis is not requiredAddedRRF bound to 70 S ribosomesRRF bound to 50 S subunits%%—100a100% binding of RRF was defined as the amount of RRF bound in the absence of EF-G (-): ∼1 pmol per pmol of 70 S ribosomes or 0.6 pmol per pmol of 50 S subunits as described under "Experimental Procedures." The reaction mixture (40 μl) contained 10 pmol of complex, and the release of RRF was measured as described under "Experimental Procedures."100a100% binding of RRF was defined as the amount of RRF bound in the absence of EF-G (-): ∼1 pmol per pmol of 70 S ribosomes or 0.6 pmol per pmol of 50 S subunits as described under "Experimental Procedures." The reaction mixture (40 μl) contained 10 pmol of complex, and the release of RRF was measured as described under "Experimental Procedures."EF-G46 ± 14NDbND, not determined.EF-G/GTP22 ± 9106 ± 8EF-G/GDP26 ± 0114 ± 14EF-G/GMPPCP10 ± 3118 ± 4a 100% binding of RRF was defined as the amount of RRF bound in the absence of EF-G (-): ∼1 pmol per pmol of 70 S ribosomes or 0.6 pmol per pmol of 50 S subunits as described under "Experimental Procedures." The reaction mixture (40 μl) contained 10 pmol of complex, and the release of RRF was measured as described under "Experimental Procedures."b ND, not determined. Open table in a new tab The fact that the release of RRF takes place with nonhydrolyzable GTP analog is very similar to the findings observed for the RRF-dependent release of tRNA catalyzed by EF-G (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). We previously suggested that the RRF-dependent release of tRNA requires translocation of RRF by EF-G. In a similar fashion, we propose that the release of RRF involves translocation of RRF from its A/P binding site to a new site by EF-G (Fig. 6). Evidence supporting this concept is given below. EF-G Does Not Release RRF from 50 S Subunits—RRF binds to 50 S subunits with a 10-fold lower affinity than to 70 S ribosomes (16Hirokawa G. Kiel M.C. Muto A. Selmer M. Raj V.S. Liljas A. Igarashi K. Kaji H. Kaji A. EMBO J. 2002; 21: 2272-2281Crossref PubMed Scopus (90) Google Scholar). In contrast to the binding of tRNA to the 30 S subunit (33Kaji H. Suzuka I. Kaji A. J. Biol. Chem. 1966; 241: 1251-1256Abstract Full Text PDF PubMed Google Scholar, 34Suzuka I. Kaji H. Kaji A. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 1483-1490Crossref PubMed Scopus (42) Google Scholar), the 30 S subunit by itself has practically no affinity for RRF (17Hirokawa G. Kiel M.C. Muto A. Kawai G. Igarashi K. Kaji H. Kaji A. J. Biol. Chem. 2002; 277: 35847-35852Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 18Lancaster L. Kiel M.C. Kaji A. Noller H.F. Cell. 2002; 111: 129-140Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), although it does play an important role in the binding of RRF to the 70 S ribosome in a similar fashion to the binding of tRNA to the 70 S ribosome (34Suzuka I. Kaji H. Kaji A. Proc. Natl. Acad. Sci. U. S. A.