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• W1986381105 abstract "During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven. During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven. Protein synthesis is a dynamic process carried out by the ribosome, an RNA-based molecular machine. During protein synthesis, tRNA and mRNA are translocated through the ribosome in a series of complex, large-scale molecular movements catalyzed by elongation factor G (EF-G) and GTP. However, translocation can occur, albeit very slowly, in the absence of EF-G and GTP (Cukras et al., 2003Cukras A.R. Southworth D.R. Brunelle J.L. Culver G.M. Green R. Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex.Mol. Cell. 2003; 12: 321-328Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, Fredrick and Noller, 2003Fredrick K. Noller H.F. Catalysis of ribosomal translocation by sparsomycin.Science. 2003; 300: 1159-1162Crossref PubMed Scopus (111) Google Scholar, Gavrilova et al., 1976Gavrilova L.P. Kostiashkina O.E. Koteliansky V.E. Rutkevitch N.M. Spirin A.S. Factor-free (“non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes.J. Mol. Biol. 1976; 101: 537-552Crossref PubMed Scopus (191) Google Scholar, Gavrilova and Spirin, 1971Gavrilova L.P. Spirin A.S. Stimulation of “non-enzymic” translocation in ribosomes by p-chloromercuribenzoate.FEBS Lett. 1971; 17: 324-326Crossref PubMed Scopus (96) Google Scholar, Pestka, 1969Pestka S. Studies on the formation of transfer ribonucleic acid-ribosome complexes. VI. Oligopeptide synthesis and translocation on ribosomes in the presence and absence of soluble transfer factors.J. Biol. Chem. 1969; 244: 1533-1539Abstract Full Text PDF PubMed Google Scholar). Thus, translocation is a property of the ribosome itself, rather than of EF-G, and is thermodynamically favored even in the absence of GTP hydrolysis. Chemical probing studies provided the first direct evidence that translocation takes place in two steps involving an intermediate hybrid state (Moazed and Noller, 1989bMoazed D. Noller H.F. Intermediate states in the movement of transfer RNA in the ribosome.Nature. 1989; 342: 142-148Crossref PubMed Scopus (588) Google Scholar). In the first step, the acceptor ends of the tRNAs move relative to the 50S subunit, from their classical A/A- and P/P-binding states into hybrid A/P and P/E states (in which the peptidyl-tRNA is bound in the 30S A site and the 50S P site and the deacylated tRNA is bound in the 30S P site and the 50S E site; Figure 1A). The specific affinity of the acceptor end of deacylated tRNA for the 50S E site (Lill et al., 1986Lill R. Robertson J.M. Wintermeyer W. Affinities of tRNA binding sites of ribosomes from Escherichia coli.Biochemistry. 1986; 25: 3245-3255Crossref PubMed Scopus (101) Google Scholar) helps to account for the thermodynamic driving force for spontaneous formation of the hybrid state. In the second step, which strongly depends on participation of EF-G, their anticodon ends move on the 30S subunit, coupled with mRNA movement, into the posttranslocational P/P and E/E states. Cryo-EM studies have identified a conformation of the ribosome in which the 30S subunit is rotated by about 3°–10° counterclockwise relative to the 50S subunit in complexes containing EF-G·GDPNP (a nonhydrolyzable analog of GTP) or EF-G·GDP·fusidic acid (Frank and Agrawal, 2000Frank J. Agrawal R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation.Nature. 2000; 406: 318-322Crossref PubMed Scopus (643) Google Scholar, Gao et al., 2003Gao H. Sengupta J. Valle M. Korostelev A. Eswar N. Stagg S.M. Van Roey P. Agrawal R.K. Harvey S.C. Sali A. et al.Study of the structural dynamics of the E. coli 70S ribosome using real-space refinement.Cell. 2003; 113: 789-801Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Valle et al., 2003Valle M. Zavialov A. Sengupta J. Rawat U. Ehrenberg M. Frank J. Locking and unlocking of ribosomal motions.Cell. 2003; 114: 123-134Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). This finding led to the proposal of a ratchet-like mechanism, in which translocation of tRNA and mRNA is linked to intersubunit rotational movement (Frank and Agrawal, 2000Frank J. Agrawal R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation.Nature. 2000; 406: 318-322Crossref PubMed Scopus (643) Google Scholar, Frank et al., 2007Frank J. Gao H. Sengupta J. Gao N. Taylor D.J. The process of mRNA-tRNA translocation.Proc. Natl. Acad. Sci. USA. 2007; 104: 19671-19678Crossref PubMed Scopus (158) Google Scholar, Tama et al., 2003Tama F. Valle M. Frank J. Brooks 3rd, C.L. Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy.Proc. Natl. Acad. Sci. USA. 2003; 100: 9319-9323Crossref PubMed Scopus (284) Google Scholar, Valle et al., 2003Valle M. Zavialov A. Sengupta J. Rawat U. Ehrenberg M. Frank J. Locking and unlocking of ribosomal motions.Cell. 2003; 114: 123-134Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Recently, this model has been directly tested by formation of a disulfide bridge between ribosomal proteins S6 and L2 designed to restrict intersubunit movement, resulting in a specific block in translocation (Horan and Noller, 2007Horan L.H. Noller H.F. Intersubunit movement is required for ribosomal translocation.Proc. Natl. Acad. Sci. USA. 2007; 104: 4881-4885Crossref PubMed Scopus (100) Google Scholar). The hybrid-state and ratchet models have now converged. Recent bulk FRET measurements combined with chemical probing experiments show that the EF-G-induced rotation of the 30S subunit observed in cryo-EM reconstructions corresponds to formation of the hybrid state characterized by chemical probing studies (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar, Ermolenko et al., 2007bErmolenko D.N. Spiegel P.C. Majumdar Z.K. Hickerson R.P. Clegg R.M. Noller H.F. The antibiotic viomycin traps the ribosome in an intermediate state of translocation.Nat. Struct. Mol. Biol. 2007; 14: 493-497Crossref PubMed Scopus (113) Google Scholar). Although EF-G binding was found to stabilize the rotated, hybrid state (Spiegel et al., 2007Spiegel P.C. Ermolenko D.N. Noller H.F. Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome.RNA. 2007; 13: 1473-1482Crossref PubMed Scopus (100) Google Scholar), rotation of the 30S subunit was also observed in the absence of EF-G under conditions favoring the hybrid state (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar), consistent with previous biochemical experiments with pretranslocation complexes (Sharma et al., 2004Sharma D. Southworth D.R. Green R. EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding.RNA. 2004; 10: 102-113Crossref PubMed Scopus (67) Google Scholar). Furthermore, spontaneous movement of two fluorescently labeled tRNAs relative to each other, interpreted as movement of the tRNAs between the classical and hybrid states, was observed in individual pretranslocation ribosomes using single-molecule FRET (smFRET) (Blanchard et al., 2004bBlanchard S.C. Kim H.D. Gonzalez Jr., R.L. Puglisi J.D. Chu S. tRNA dynamics on the ribosome during translation.Proc. Natl. Acad. Sci. USA. 2004; 101: 12893-12898Crossref PubMed Scopus (354) Google Scholar, Kim et al., 2007Kim H.D. Puglisi J. Chu S. Fluctuations of tRNAs between classical and hybrid states.Biophys. J. 2007; 104: 13661-13665Google Scholar, Munro et al., 2007Munro J.B. Altman R.B. O'Connor N. Blanchard S.C. Identification of two distinct hybrid state intermediates on the ribosome.Mol. Cell. 2007; 25: 505-517Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Although the above-mentioned evidence points to the role of ribosome structural dynamics in translocation, the underlying molecular mechanism of this process remains elusive. Intersubunit movements inferred from cryo-EM and static bulk FRET experiments have been performed at equilibrium and on the ensemble level and have yet to be observed in real time; moreover, there is so far no thermodynamic and kinetic description of ribosomal intersubunit movement. Finally, the proposal, based on cryo-EM (Frank and Agrawal, 2000Frank J. Agrawal R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation.Nature. 2000; 406: 318-322Crossref PubMed Scopus (643) Google Scholar, Gao et al., 2004Gao H. Valle M. Ehrenberg M. Frank J. Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset.J. Struct. Biol. 2004; 147: 283-290Crossref PubMed Scopus (69) Google Scholar) and FRET studies (Munro et al., 2007Munro J.B. Altman R.B. O'Connor N. Blanchard S.C. Identification of two distinct hybrid state intermediates on the ribosome.Mol. Cell. 2007; 25: 505-517Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, Pan et al., 2007Pan D. Kirillov S.V. Cooperman B.S. Kinetically competent intermediates in the translocation step of protein synthesis.Mol. Cell. 2007; 25: 519-529Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), that ribosomal subunits may occupy more than one intermediate conformational state has yet to be established. Here, we address these questions directly using smFRET (Ha et al., 1996Ha T. Enderle T. Ogletree D.F. Chemla D.S. Selvin P.R. Weiss S. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.Proc. Natl. Acad. Sci. USA. 1996; 93: 6264-6268Crossref PubMed Scopus (922) Google Scholar) and total internal reflection microscopy (Zhuang et al., 2000Zhuang X. Bartley L.E. Babcock H.P. Russell R. Ha T. Herschlag D. Chu S. A single-molecule study of RNA catalysis and folding.Science. 2000; 288: 2048-2051Crossref PubMed Scopus (622) Google Scholar). This method has been used previously to probe tRNA dynamics during and after tRNA accommodation (Blanchard et al., 2004aBlanchard S.C. Gonzalez R.L. Kim H.D. Chu S. Puglisi J.D. tRNA selection and kinetic proofreading in translation.Nat. Struct. Mol. Biol. 2004; 11: 1008-1014Crossref PubMed Scopus (359) Google Scholar, Blanchard et al., 2004bBlanchard S.C. Kim H.D. Gonzalez Jr., R.L. Puglisi J.D. Chu S. tRNA dynamics on the ribosome during translation.Proc. Natl. Acad. Sci. USA. 2004; 101: 12893-12898Crossref PubMed Scopus (354) Google Scholar, Gonzalez et al., 2007Gonzalez Jr., R.L. Chu S. Puglisi J.D. Thiostrepton inhibition of tRNA delivery to the ribosome.RNA. 2007; 13: 2091-2097Crossref PubMed Scopus (42) Google Scholar, Kim et al., 2007Kim H.D. Puglisi J. Chu S. Fluctuations of tRNAs between classical and hybrid states.Biophys. J. 2007; 104: 13661-13665Google Scholar, Lee et al., 2007Lee T.H. Blanchard S.C. Kim H.D. Puglisi J.D. Chu S. The role of fluctuations in tRNA selection by the ribosome.Proc. Natl. Acad. Sci. USA. 2007; 104: 13661-13665Crossref PubMed Scopus (82) Google Scholar, Munro et al., 2007Munro J.B. Altman R.B. O'Connor N. Blanchard S.C. Identification of two distinct hybrid state intermediates on the ribosome.Mol. Cell. 2007; 25: 505-517Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and EF-G dynamics (Wang et al., 2007Wang Y. Qin H. Kudaravalli R.D. Kirillov S.V. Dempsey G.T. Pan D. Cooperman B.S. Goldman Y.E. Single-molecule structural dynamics of EF-G-ribosome interaction during translocation.Biochemistry. 2007; 46: 10767-10775Crossref PubMed Scopus (51) Google Scholar) on the ribosome. In our experiments, using fluorescently labeled ribosomal subunits, we use this approach to directly monitor the dynamics of the ribosome itself. We observe the hypothesized ratchet-like motions of individual ribosomes and characterize the determining factors of their dynamics. The ability of ribosomes to undergo spontaneous intersubunit rotation in the absence of EF-G or GTP has strong implications for the molecular mechanism of translocation. We conjugated fluorescent labels to specific cysteine residues introduced by directed mutagenesis into ribosomal proteins S6 (D41C), S11 (E75C), and L9 (N11C) (Figure 1B) (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar, Ermolenko et al., 2007bErmolenko D.N. Spiegel P.C. Majumdar Z.K. Hickerson R.P. Clegg R.M. Noller H.F. The antibiotic viomycin traps the ribosome in an intermediate state of translocation.Nat. Struct. Mol. Biol. 2007; 14: 493-497Crossref PubMed Scopus (113) Google Scholar). Proteins S6 or S11 labeled with acceptor (Cy5) dye and protein L9 labeled with donor (Cy3) dye were incorporated into 30S and 50S subunits, respectively, by in vitro reconstitution as described previously (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar). The labeled subunits were then associated to create two kinds of doubly labeled 70S ribosomes, 70S:S6(Cy5)/L9(Cy3) and 70S:S11(Cy5)/L9(Cy3). In vitro assays showed that at least 50%–60% of purified reconstituted, labeled ribosomes were active in in vitro translocation (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar) and 80%–100% active in in vitro translation of a defined mRNA (L. Lancaster, personal communication). In order to study the intrinsic structural dynamics of the pretranslocation ribosome, the peptidyl-tRNA analog N-Ac-Phe-tRNAPhe was bound to the A site of ribosomes containing deacylated tRNAfMet bound to the P site in the presence of a defined mRNA. Pretranslocation ribosome complexes were then immobilized in polymer-passivated microscope slide/coverslip chambers via a biotin-derivatized DNA oligonucleotide annealed to the mRNA (Figure 1C) (Blanchard et al., 2004bBlanchard S.C. Kim H.D. Gonzalez Jr., R.L. Puglisi J.D. Chu S. tRNA dynamics on the ribosome during translation.Proc. Natl. Acad. Sci. USA. 2004; 101: 12893-12898Crossref PubMed Scopus (354) Google Scholar) and were visualized using total internal reflection microscopy (Zhuang et al., 2000Zhuang X. Bartley L.E. Babcock H.P. Russell R. Ha T. Herschlag D. Chu S. A single-molecule study of RNA catalysis and folding.Science. 2000; 288: 2048-2051Crossref PubMed Scopus (622) Google Scholar). This immobilization approach preserved the ribosome's translocation activity (see below). Time traces of individual S6-Cy5/L9-Cy3 pretranslocation complexes showed spontaneous, time-dependent anticorrelated changes in donor (Cy3) and acceptor (Cy5) fluorescence intensities (Figure 2A). Calculation of apparent FRET efficiency (FRET = ICy5/[ICy5 + ICy3]) from donor (ICy3) and acceptor (ICy5) intensities revealed that pretranslocation ribosomes fluctuate between high (0.56) and low (0.40) FRET states. smFRET measurements performed with the Cy3 and Cy5 dyes reversed gave similar results (data not shown). Time traces recorded for S11-Cy5/L9-Cy3 ribosomes show a similar pattern of spontaneous fluctuations but inverted from that of the S6/L9 construct (data not shown), because S11 moves closer to L9 in the hybrid state, whereas S6 moves away from L9 (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar). Below, we present only data from the S6/L9 construct, because of the previously demonstrated strong anticorrelation between the FRET changes for the S6/L9 and S11/L9 dye pairs (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar). The high (0.56) FRET state for the S6/L9 pair corresponds to the nonrotated conformation of the ribosome, in which the tRNAs are bound in the classical state, whereas the low (0.4) FRET state corresponds to the conformation in which the small ribosomal subunit is rotated and the tRNAs are bound in the A/P and P/E hybrid states (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar, Ermolenko et al., 2007bErmolenko D.N. Spiegel P.C. Majumdar Z.K. Hickerson R.P. Clegg R.M. Noller H.F. The antibiotic viomycin traps the ribosome in an intermediate state of translocation.Nat. Struct. Mol. Biol. 2007; 14: 493-497Crossref PubMed Scopus (113) Google Scholar). The latter conformation is equivalent to the “ratcheted state” observed in cryo-EM studies of complexes of the ribosome bound with EF-G (Frank and Agrawal, 2000Frank J. Agrawal R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation.Nature. 2000; 406: 318-322Crossref PubMed Scopus (643) Google Scholar, Valle et al., 2003Valle M. Zavialov A. Sengupta J. Rawat U. Ehrenberg M. Frank J. Locking and unlocking of ribosomal motions.Cell. 2003; 114: 123-134Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Thus, our single-ribosome traces show that the pretranslocation ribosome, in the absence of EF-G or GTP, fluctuates spontaneously between the rotated and nonrotated conformations, corresponding to the hybrid and classical states, respectively. To ask whether the ribosomal subunits also move through any additional, previously unobserved transient rotational states, the presence of which could be masked by noise in our FRET traces, we subjected our data to a hidden Markov modeling (HMM) algorithm (McKinney et al., 2006McKinney S.A. Joo C. Ha T. Analysis of single-molecule FRET trajectories using hidden Markov modeling.Biophys. J. 2006; 91: 1941-1951Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). This approach allows for determination of the number of states present in the system and the rates of exchange between them. HMM analysis of the S6-Cy5/L9-Cy3 pre-translocation complex (612 total molecules showing on average 30 transitions per molecule; Table 1) assuming three states (Figures 2C and 3) showed that the pretranslocation complex fluctuates between just two distinct states, nonrotated and rotated, with a forward rotation (nonrotated to rotated) rate of 0.27 ± 0.08 s−1 and a reverse rotation (rotated to nonrotated) rate of 0.19 ± 0.02 s−1 at 25°C under our in vitro conditions (Figures 2B and 2C; Table 1).Table 1Kinetic Rates Measured between 0.56 and 0.40 FRET StatesP Site tRNA/A site tRNAForward Transitions (k1)k1(s−1)Reverse Transitions (kminus1)kminus1(s−1)TransitIons per TracetRNAfMet/Vacant99060.51 ± 0.0399020.21 ± 0.0335tRNAfMet/N-Ac-Phe-tRNAPhe92560.27 ± 0.0891950.19 ± 0.0230tRNAMet/Vacant32560.49 ± 0.1232420.09 ± 0.0317Results of fitting FRET time trajectories with the HMM algorithm. Each data set was divided into three and analyzed separately. The reported number is an average from each of the three data sets with the standard deviation. Open table in a new tab Figure 3Representative Time Traces from HMM Analysis of S6-Cy5/L9-Cy3 Ribosomes Containing Deacylated tRNAfMet in the P Site and N-Ac-Phe-tRNAPhe in the A SiteShow full captionCy3 and Cy5 intensities are shown as green and red traces, respectively. The calculated FRET curve is shown in blue with the HMM-determined fit overlaid in black.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Results of fitting FRET time trajectories with the HMM algorithm. Each data set was divided into three and analyzed separately. The reported number is an average from each of the three data sets with the standard deviation. Cy3 and Cy5 intensities are shown as green and red traces, respectively. The calculated FRET curve is shown in blue with the HMM-determined fit overlaid in black. The same analysis performed on ribosomes containing only a P site tRNA (tRNAfMet or tRNAMet) also resulted in just two observed FRET states (Table 1 and see Figure S1 available with this article online). In addition, dwell-time analysis for all three complexes fit to a single exponential decay, consistent with two states (Figure S2). We next asked how the acylation state of P site tRNA and EF-G binding affects the nonrotated/rotated states' equilibrium. The equilibrium constant, Keq=[%rotated]/[%non−rotated], is determined from double Gaussian fits to smFRET histograms (Dahan et al., 1999Dahan M. Deniz A.A. Ha T. Chemla D.S. Schultz P.G. Weiss S. Ratiometric measurement and identification of single diffusing molecules.Chem. Phys. 1999; 247: 85-106Crossref Scopus (131) Google Scholar) built from several hundred molecules for each construct (Figure 4 and Figure S3; Table 2). A majority (75%) of posttranslocation ribosomes, containing the peptidyl-tRNA analog N-Ac-Phe-tRNAPhe bound in the P site with a vacant A site, exhibited a high FRET value (Figure 4A), corresponding to the classical, nonrotated conformation, in agreement with chemical probing (Moazed and Noller, 1989bMoazed D. Noller H.F. Intermediate states in the movement of transfer RNA in the ribosome.Nature. 1989; 342: 142-148Crossref PubMed Scopus (588) Google Scholar) and bulk FRET (Ermolenko et al., 2007aErmolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. Observation of intersubunit movement of the ribosome in solution using FRET.J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (142) Google Scholar) experiments. In a complex containing a different peptidyl tRNA, fMet-tRNAfMet, bound to the P site, 66% of ribosomes were also in the nonrotated conformation (Figure 4B). Likewise, 52% and 79% of authentic posttranslocation complexes obtained by incubation of the pretranslocation complex (the former containing the N-Ac-Phe-tRNAPhe bound to the A site and deacylated tRNAfMet bound to the P site and the latter containing N-Ac-Phe-tRNAPhe bound to the A site and deacylated tRNATyr bound to the P site) with EF-G·GTP were found in the nonrotated conformation (Figures 4C and 4D). Sixty-one percent of vacant ribosomes (i.e., ones lacking tRNA) also exhibited a high FRET value (Figure 4K).Table 2Statistical Data for All Tested ComplexesFigurePercent NRPercent RKeqk1 (s−1)k−1 (s−1)MethodPercent TransVacant and ASL in P SiteVacant4K61aValues in this column are derived from fitting the histograms providing the percent of the molecules in the nonrotated (NR) or high FRET state and the rotated (R) or low FRET state.39aValues in this column are derived from fitting the histograms providing the percent of the molecules in the nonrotated (NR) or high FRET state and the rotated (R) or low FRET state.0.63bEquilibrium constants in this column are calculated from the relative populations of nonrotated and rotated ribosomes (see text).0.015cThe rates in this column were calculated as described in the text and in the Experimental Procedures.0.020cThe rates in this column were calculated as described in the text and in the Experimental Procedures.1, 1dIn this column, the method used to calculate the rates in which 1 is the number of transitions/dwell time, 2 is HMM analysis, and 3 is dwell-time analysis. A more detailed description is in the Experimental Procedures.4eThe percentages in this column represent the total number of traces that contain at least one unambiguous FRET transition between the fitted high and low FRET states divided by the total number of traces.Vacant with EF-G·GDPNP4O49511.10.0040.0031, 15ASLfMet4L71290.410.0060.0051, 13ASLfMet with EF-G·GDPNP4P71290.410.0040.0091, 12Peptidyl-tRNA in P SiteN-Ac-Phe-tRNAPhe4A75250.330.0050.0161, 14fMet-tRNAfMet4B66340.500.0060.0131, 12Pretranslocation ComplexestRNAfMet/N-Ac-Phe-tRNAPhe4G31692.20.27 ± 0.080.19 ± 0.022, 259tRNATyr/N-Ac-Phe-tRNAPhe4H15855.71.65 ± 0.120.0193, 114Posttranslocation ComplexestRNAfMet/N-Ac-Phe-tRNAPhe with EF-G·GTP4C52480.920.0340.0311, 18tRNATyr/N-Ac-Phe-tRNAPhe with EF-G·GTP4D79210.270.0200.0741, 19Deacyl-tRNA in P SitetRNAfMet4F23773.30.51 ± 0.030.21 ± 0.032, 271tRNAfMet with EF-G·GDPNP4J694171.19 ± 0.040.0213, 17tRNAfMet viomycin4N10909.2NDND3tRNAPhe4E15855.61.14 ± 0.010.26 ± 0.013, 345tRNATyr4M892122.53 ± 0.340.0243, 112tRNATyr with EF-G·GDPNPS3B0100-NDND1tRNATyr with viomycinS3D11898.5NDND0tRNAMet4I15855.60.49 ± 0.120.09 ± 0.032, 251tRNAMet with EF-G·GDPNPS3A99110NDND3tRNAMet with viomycinS3C0100-NDND0ND, insufficient data to calculate rate information. For complexes stabilized in the rotated conformation (e.g., complexes containing EF-G or viomycin), equilibrium constants calculated from the relative populations of nonrotated and rotated ribosomes deviate significantly from constants calculated from the ratios of rates for forward and reverse rotation. This discrepancy is likely due to the presence of a fraction of inactive ribosomes that skews the value of the equilibrium constant calculated from the distribution.a Values in this column are derived from fitting the histograms providing the percent of the molecules in the nonrotated (NR) or high FRET state and the rotated (R) or low FRET state.b Equilibrium constants in this column are calculated from the relative populations of nonrotated and rotated ribosomes (see text).c The rates in this column were calculated as described in the text and in the Experimental Procedures.d In this column, the method used to calculate the rates in which 1 is the number of transitions/dwell time, 2 is HMM analysis, and 3 is dwell-time analysis. A more detailed description is in the Experimental Procedures.e The percentages in this column represent the total number of traces that contain at least one unambiguous FRET transition between the fitted high and low FRET states divided by the total number of traces. Open table in a new tab ND, insufficient data to calculate rate information. For complexes stabilized in the rotated conformation (e.g., complexes containing EF-G or viomycin), equilibrium constants calculated from the relative populations of nonrotated and rotated ribosomes deviate significantly from constants calculated from the ratios of rates for forward and reverse rotation. This discrepancy is likely due to the presence of a fraction of inactive ribosomes that skews the value of the equilibrium constant calculated from the distribution. In contrast to posttranslocation complexes, ribosomes containing deacylated tRNAfMet in the P site without an A site tRNA showed a majority of ribosomes (77%) in the low-FRET rotated s" @default.
• W1986381105 created "2016-06-24" @default.
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