Bridging Conformational Dynamics and Function Using Single-Molecule Spectroscopy
2006; Elsevier BV; Volume: 14; Issue: 4 Linguagem: Inglês
10.1016/j.str.2006.02.005
ISSN1878-4186
AutoresSua Myong, Benjamin Stevens, Taekjip Ha,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoIn a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the "structure-function relation" by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase. In a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the "structure-function relation" by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase. There is compelling need for new tools to probe the relationship between structure and function of biological macromolecules. Whereas high-resolution structural tools such as X-ray crystallography and nuclear magnetic resonance provide snapshots at atomic detail, they do not always tell us what the molecule does and how it does it. Biochemical techniques provide valuable functional information, and in combination, these structural and biochemical tools have helped us deepen our understanding of living systems at the molecular level. Ideally, one would like to observe the structural dynamics and function simultaneously so that a direct correlation can be made between the two. Single-molecule fluorescence spectroscopy has proven to be up to this challenge by measuring the real-time conformational changes of individual molecules of DNA, RNA, and protein during their function. Single-molecule studies have also revealed that nominally identical molecules can behave very differently from each other even under almost identical conditions. Such heterogeneity used to garner much interest and is fascinating in its own right, but for most cases its functional significance was unclear and strong proof that it was not a measurement artifact was lacking. Both technical improvements and closer ties to biology led to a flurry of recent studies which go well beyond the sort pronouncing that "the most interesting observation made in these first single-molecule experiments on System X is that single molecules are very heterogeneous!" This review is about a small collection of these studies that exhibit the virtue of single-molecule spectroscopy in providing previously unattainable data on biological mechanisms. In the first two sections on the hairpin ribozyme and Holliday junction, two-state fluctuations were observed due to docking/undocking reactions and stacking conformer dynamics, respectively. Interestingly, their "ticking" rates were not constant during the measurements but switched between two different sets of values. It is as though the molecule has two internal clocks that run at different speeds and it uses one clock for a while and then switches to the other and back, and it goes on. This so-called memory effect, where a molecule seems to remember which clock to use, was first reported in single-molecule fluorescence studies on the enzymatic turnover of cholesterol oxidase (Lu et al., 1998Lu H.P. Xun L.Y. Xie X.S. Single-molecule enzymatic dynamics.Science. 1998; 282: 1877-1882Crossref PubMed Google Scholar). In the new study reviewed here, the underlying mechanisms of switching between the clocks were unambiguously shown: cleavage and ligation reactions for the hairpin ribozyme (Nahas et al., 2004Nahas M.K. Wilson T.J. Hohng S. Lilley D.M.J. Ha T. Observation of internal cleavage and ligation reactions of a ribozyme.Nat. Struct. Mol. Biol. 2004; 11: 1107-1113Crossref PubMed Scopus (88) Google Scholar) and single-step branch migration for the Holliday junction (McKinney et al., 2005McKinney S.A. Freeman A.D. Lilley D.M. Ha T. Observing spontaneous branch migration of Holliday junctions one step at a time.Proc. Natl. Acad. Sci. USA. 2005; 102: 5715-5720Crossref PubMed Scopus (68) Google Scholar). The third section reviews the tRNA dynamics in the ribosome where transient intermediates toward tRNA accommodation were clearly observed for the first time (Blanchard et al., 2004Blanchard 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., 2004Blanchard 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). The fourth section concerns the ATP-powered movement of a helicase protein on a single-stranded DNA (ssDNA). When the protein encountered an insurmountable obstacle, it was observed to employ a series of acrobatic moves to return to where it began and repeat the process over and over again, an unexpected finding which points to a potential new function of keeping ssDNA clear of unwanted proteins (Myong et al., 2005Myong S. Rasnik I. Joo C. Lohman T.M. Ha T. Repetitive shuttling of a motor protein on DNA.Nature. 2005; 437: 1321-1325Crossref PubMed Scopus (213) Google Scholar). The final section is about the direct measurement of discrete rotational steps of a protein complex called F0F1-ATP synthase during ATP synthesis (Diez et al., 2004Diez M. Zimmermann B. Borsch M. Konig M. Schweinberger E. Steigmiller S. Reuter R. Felekyan S. Kudryavtsev V. Seidel C.A. Graber P. Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.Nat. Struct. Mol. Biol. 2004; 11: 135-141Crossref PubMed Scopus (334) Google Scholar, Zimmermann et al., 2005Zimmermann B. Diez M. Zarrabi N. Graber P. Borsch M. Movements of the ɛ-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase.EMBO J. 2005; 24: 2053-2063Crossref PubMed Scopus (96) Google Scholar). Both the direction of rotation (clockwise versus anticlockwise) and the asymmetry in dwell times for three different rotor angles could be determined. In all five studies, the primary signal was single-molecule fluorescence resonance energy transfer (FRET) (Ha, 2001Ha T. Single-molecule fluorescence resonance energy transfer.Methods. 2001; 25: 78-86Crossref PubMed Scopus (459) Google Scholar), an excellent tool for measuring relative distance changes between two fluorophores, donor and acceptor, attached to specific sites on the macromolecules (Weiss, 1999Weiss S. Fluorescence spectroscopy of single biomolecules.Science. 1999; 283: 1676-1683Crossref PubMed Scopus (1768) Google Scholar). It should be noted, however, that there are other observables in single-molecule fluorescence measurements, notably intensity/lifetime (Laurence et al., 2005Laurence T.A. Kong X. Jager M. Weiss S. Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins.Proc. Natl. Acad. Sci. USA. 2005; 102: 17348-17353Crossref PubMed Scopus (186) Google Scholar, Lu et al., 1998Lu H.P. Xun L.Y. Xie X.S. Single-molecule enzymatic dynamics.Science. 1998; 282: 1877-1882Crossref PubMed Google Scholar, Yang et al., 2003Yang H. Luo G. Karnchanaphanurach P. Louie T.M. Rech I. Cova S. Xun L. Xie X.S. Protein conformational dynamics probed by single-molecule electron transfer.Science. 2003; 302: 262-266Crossref PubMed Scopus (729) Google Scholar) and polarization response (Forkey et al., 2003Forkey J.N. Quinlan M.E. Shaw M.A. Corrie J.E. Goldman Y.E. Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization.Nature. 2003; 422: 399-404Crossref PubMed Scopus (357) Google Scholar, Nishizaka et al., 2004Nishizaka T. Oiwa K. Noji H. Kimura S. Muneyuki E. Yoshida M. Kinosita Jr., K. Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.Nat. Struct. Mol. Biol. 2004; 11: 142-148Crossref PubMed Scopus (232) Google Scholar, Sosa et al., 2001Sosa H. Peterman E.J.G. Moerner W.E. Goldstein L.S.B. ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy.Nat. Struct. Biol. 2001; 8: 540-544Crossref PubMed Scopus (134) Google Scholar). The hairpin ribozyme is an RNA enzyme that carries out site-specific self-cleavage and ligation of viral RNA. The ribozyme has modular architecture built of a four-way junction and two adjacent loops on the two arms of the junction (Figure 1A). The catalytic site is formed by docking the two loops onto each other, stabilized by divalent metal ions. The four-way junction is considered a folding enhancer because docking is accelerated 500-fold due to the junction-derived fluctuations within the undocked state that brings the two loops close to each other repeatedly (Tan et al., 2003Tan E. Wilson T.J. Nahas M.K. Clegg R.M. Lilley D.M.J. Ha T. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate.Proc. Natl. Acad. Sci. USA. 2003; 100: 9308-9313Crossref PubMed Scopus (172) Google Scholar). In the single-molecule studies, the ribozyme is labeled with a donor and an acceptor at the ends of the two loop-carrying arms so that the high FRET and low FRET signals would represent the docked and undocked states, respectively (Figure 1A). Cleavage and ligation would occur only within the high FRET docked state. In typical biochemical studies of the cleavage reaction, the product RNA piece once cleaved from the ribozyme no longer binds to the ribozyme tightly and quickly dissociates, effectively rendering the reaction irreversible. In order to observe multiple cleavage/ligation cycles, Nahas et al., 2004Nahas M.K. Wilson T.J. Hohng S. Lilley D.M.J. Ha T. Observation of internal cleavage and ligation reactions of a ribozyme.Nat. Struct. Mol. Biol. 2004; 11: 1107-1113Crossref PubMed Scopus (88) Google Scholar lengthened the RNA piece, which allows the cleavage product to remain bound through 7 bp. At 1 mM magnesium, most molecules were found in a stably docked state (high FRET) that was interspersed with bursts of rapid docking and undocking (Figure 1B). These bursts were a striking contrast to the steady high FRET signal obtained from the noncleavable mutant (Tan et al., 2003Tan E. Wilson T.J. Nahas M.K. Clegg R.M. Lilley D.M.J. Ha T. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate.Proc. Natl. Acad. Sci. USA. 2003; 100: 9308-9313Crossref PubMed Scopus (172) Google Scholar). From this comparison it was hypothesized that the stably docked form is the ligated ribozyme and that the rapidly fluctuating form corresponds to the cleaved ribozyme. To test this hypothesis, ribozymes that had not been previously exposed to magnesium ions (and should be in the ligated form) were immobilized onto a quartz surface (Figure 2A). When magnesium ions were added via flow, the ribozymes went from an undocked state to a stably docked state (Figure 2B). Only later were rapid fluctuations observed, likely as a result of cleavage. Therefore, the stably docked form was assigned to the ligated ribozyme. In the second control experiment, ribozymes which initially had a short cleavage substrate were exposed to magnesium, resulting in a cleavage reaction and quick dissociation of the product. This left the ribozyme with an impaired loop structure which is unable to fold and hence exhibited a low FRET signal. These ribozymes were observed while a buffer containing a high concentration of long (7 bp) cleavage product was flowed in. When this long ligatable product strand bound to the ribozyme, a switch from low FRET to rapidly fluctuating behavior was observed followed by stable high FRET (Figures 2C and 2D). Because the ribozyme must be in the cleaved form immediately after the long product strand binds, the rapidly fluctuating FRET signal was assigned to the ribozyme in the cleaved form. Thus, it is now possible to record the exact moment when the cleavage or ligation reaction occurs via the changes in the docking/undocking kinetics (marked by inverted triangles in Figure 1, Figure 2). This opens up exciting new opportunities to carry out single-molecule enzymology in the most direct way, where the effects of mutations or solution conditions on the internal chemistry can be examined without complications arising from conformational degrees of freedom. For example, Nahas et al. determined the rates of internal cleavage and ligation reactions as a function of pH (Nahas et al., 2004Nahas M.K. Wilson T.J. Hohng S. Lilley D.M.J. Ha T. Observation of internal cleavage and ligation reactions of a ribozyme.Nat. Struct. Mol. Biol. 2004; 11: 1107-1113Crossref PubMed Scopus (88) Google Scholar), which provides support for general acid-base catalysis (Lilley, 2005Lilley D.M. Structure, folding and mechanisms of ribozymes.Curr. Opin. Struct. Biol. 2005; 15: 313-323Crossref PubMed Scopus (123) Google Scholar). Why is undocking accelerated so much upon cleavage? Nahas et al., 2004Nahas M.K. Wilson T.J. Hohng S. Lilley D.M.J. Ha T. Observation of internal cleavage and ligation reactions of a ribozyme.Nat. Struct. Mol. Biol. 2004; 11: 1107-1113Crossref PubMed Scopus (88) Google Scholar speculated that rapid undocking after cleavage may be used to present the cleavage product for the next step in the replication of the viral RNA instead of allowing religation to occur. Surprisingly, such acceleration was not observed if the cleavage product contained a nonnatural terminus (Zhuang et al., 2002Zhuang X.W. Kim H. Pereira M.J.B. Babcock H.P. Walter N.G. Chu S. Correlating structural dynamics and function in single ribozyme molecules.Science. 2002; 296: 1473-1476Crossref PubMed Scopus (422) Google Scholar), a commonly used approach to approximate the cleaved ribozyme. Thus, it has to be recognized that alterations to the hairpin ribozyme (and possibly other ribozymes) can affect both folding and catalytic properties, and the ability to probe the internal chemistry directly as demonstrated by Nahas et al., 2004Nahas M.K. Wilson T.J. Hohng S. Lilley D.M.J. Ha T. Observation of internal cleavage and ligation reactions of a ribozyme.Nat. Struct. Mol. Biol. 2004; 11: 1107-1113Crossref PubMed Scopus (88) Google Scholar should be valuable. The power of single-molecule FRET spectroscopy in RNA folding and catalysis studies has been demonstrated in a number of systems (Bokinsky et al., 2003Bokinsky G. Rueda D. Misra V.K. Rhodes M.M. Gordus A. Babcock H.P. Walter N.G. Zhuang X. Single-molecule transition-state analysis of RNA folding.Proc. Natl. Acad. Sci. USA. 2003; 100: 9302-9307Crossref PubMed Scopus (166) Google Scholar, Ha et al., 1999Ha T. Zhuang X.W. Kim H.D. Orr J.W. Williamson J.R. Chu S. 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The Holliday junction is formed when two DNA molecules exchange strands during homologous recombination, an important process in maintaining genome stability and diversity. These junctions are capable of spontaneous branch migration because of sequence homology, but almost all structural studies have so far focused on nonhomologous junctions whose sequences forbid branch migration. In the absence of metal ions, the junction adopts an "open structure," where the four helices are pointing to four corners of a square. In the presence of divalent metal ions, the junction folds into a more compact "stacked X-structure" (Murchie et al., 1989Murchie A.I. Clegg R.M. von Kitzing E. Duckett D.R. Diekmann S. Lilley D.M. Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules.Nature. 1989; 341: 763-766Crossref PubMed Scopus (314) Google Scholar), where two possible conformers are in continual exchange through the open intermediate (Joo et al., 2004Joo C. McKinney S.A. Lilley D.M.J. Ha T. Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy.J. Mol. Biol. 2004; 341: 739-751Crossref PubMed Scopus (83) Google Scholar, McKinney et al., 2003McKinney S.A. Declais A.C. Lilley D.M.J. Ha T. Structural dynamics of individual Holliday junctions.Nat. Struct. Biol. 2003; 10: 93-97Crossref PubMed Scopus (256) Google Scholar, Miick et al., 1997Miick S.M. Fee R.S. Millar D.P. Chazin W.J. Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions.Proc. Natl. Acad. Sci. 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Lilley D.M. Ha T. Observing spontaneous branch migration of Holliday junctions one step at a time.Proc. Natl. Acad. Sci. USA. 2005; 102: 5715-5720Crossref PubMed Scopus (68) Google Scholar designed the monomigratable junction, whose sequence allows only a single step of branch migration between two branch points termed U and M (Figure 3A). The strands were labeled with two fluorophores attached to the ends of the two adjacent arms that would show low and high FRET differentiating two stacking conformations, isoI and isoII, respectively (Figure 3A). Single-molecule traces displayed fluctuations between four states which could be grouped into two phases, slow- and fast-fluctuating (Figure 3B). Single molecules were observed to switch between the two distinct phases, and the switching was interpreted as the moment branch migration occurred (vertical arrows in Figure 3B) because such behavior was never observed in junctions with a fixed branch point. Hydroxyl radical cleavage probing confirmed this interpretation and assigned the two phases to definite locations of the branch point (slow phase for U and fast phase for M; Figures 3A and 3B). Adopting a physical metaphor, the junction can be thought of as a pendulum with a defined frequency, and branch migration as a spring linking two pendulums. If one pendulum is induced to swing, the energy eventually flows into the second pendulum and back and so on. If the coupling is weak, each pendulum swings multiple times, more or less behaving as an independent pendulum, until eventually energy sloshes to the other one. It turns out that the monomigratable junctions studied here retained within each branch point the hallmark behavior of nonhomologous junctions: two-state fluctuations whose overall rates, but not their relative populations, depend on magnesium concentrations. Thus, we can conclude that "migrability" is a weak perturbation. On average, the junctions displayed approximately ten oscillations before branch migration but the exact number depended on whether GC versus AT base pairs are broken during branch migration. One surprise was that the different branch points could have up to 30-fold variations in their lifetimes, as illustrated in Figure 3C. Thus, spontaneous branch migration may occur very rapidly in a local segment bounded by very stable branch points. A similar conclusion was also reached by a recent single-molecule study of junctions that can migrate over several different branch points (Karymov et al., 2005Karymov M. Daniel D. Sankey O.F. Lyubchenko Y.L. Holliday junction dynamics and branch migration: single-molecule analysis.Proc. Natl. Acad. Sci. USA. 2005; 102: 8186-8191Crossref PubMed Scopus (42) Google Scholar). In vivo, junctions can quickly find stable branch points if they are transiently free of protein, possibly affecting the outcome of homologous recombination. The new knowledge on the dynamic properties of the naked junction should help design and interpret future studies of enzymes that recognize and process the Holliday junction. Ribosomes make protein using mRNA as a template. In bacteria, the ribosome is made of two subunits, 30S and 50S (Figure 4). The mRNA binds to the 30S such that the codon in mRNA is recognized by base pairing to the anticodon in the tRNA that brings an amino acid to be linked to the growing peptide. Using single-molecule FRET between tRNA molecules in the aminoacyl (A) and peptidyl (P) sites (Figure 4), Blanchard et al., 2004Blanchard 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 probed the mechanism for the very high translation fidelity which cannot be explained solely by codon/anticodon binding energy differences between the cognate and noncognate tRNA (Rodnina and Wintermeyer, 2001Rodnina M.V. Wintermeyer W. Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms.Annu. Rev. Biochem. 2001; 70: 415-435Crossref PubMed Scopus (229) Google Scholar). Ribosome complexes that contain a donor-labeled tRNA at the P site were formed on biotinylated mRNA (Blanchard et al., 2004Blanchard 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) that had been specifically bound to a passivated, PEG-covered surface (Ha et al., 2002Ha T. Rasnik I. Cheng W. Babcock H.P. Gauss G. Lohman T.M. Chu S. Initiation and reinitiation of DNA unwinding by the Escherichia coli Rep helicase.Nature. 2002; 419: 638-641Crossref PubMed Scopus (370) Google Scholar, Rasnik et al., 2004Rasnik I. Myong S. Cheng W. Lohman T.M. Ha T. DNA-binding orientation and domain conformation of the E. coli Rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes.J. Mol. Biol. 2004; 336: 395-408Crossref PubMed Scopus (130) Google Scholar; Figure 4). Then, ternary complexes containing an acceptor-labeled aminoacyl tRNA, EF-Tu (a GTPase), and GTP were flowed into the sample as single-molecule FRET trajectories were being recorded during the accommodation of the ternary complex at the A site. Because the moment of the ternary complex binding varied between single-molecule traces, a postsynchronization technique was employed wherein many FRET traces were synchronized at the first time FRET efficiency went above an established noise threshold of 0.25 (Figure 5A; Blanchard et al., 2004Blanchard 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). For cognate tRNAs, single-molecule traces transited through low and intermediate FRET (0.3–0.5) to high FRET (0.6 and above) as the ternary complex accommodated into the A site. The antibiotic tetracycline was found to allow initial binding and codon recognition, but not full accommodation; a short-lived FRET state of 0.35 was observed. Replacing GTP with a nonhydrolyzable analog GDPNP stalled the complex in a longer lived (∼8 s) 0.5 FRET state, indicating the formation of more contacts between the ternary complex and the ribosome. Cleavage of the sarcin-ricin loop, part of the GTPase activating center (GAC), known to play a role in tRNA selection, also stalled the reaction at the 0.5 FRET state. Near-cognate (one-base mismatch) tRNAs tend to dissociate from the ribosome from the low and intermediate FRET states rather than progressing further in accommodation. The ratio of probabilities with which cognate and near-cognate tRNA progressed from low to intermediate FRET and then intermediate to high FRET was compared to assess the fidelity of the system. This analysis revealed that the two previously established proofreading steps in tRNA selection corresponded to the branch points in the FRET data; cognate tRNA tends to progress through these branch points, whereas near-cognate tRNA tends to dissociate from the ribosome. Thus, initial selection and proofreading steps could be directly related to FRET transitions and therefore tRNA movements within the A site. These data were used to estimate the fidelity of tRNA selection at roughly 1 miscoding error in 5000. Based on these data, a model of tRNA selection was proposed (Blanchard et al., 2004Blanchard 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; Figure 5C). In step 0, the P site is occupied by peptidyl-tRNA. In step 1, the ternary complex approaches the ribosome and FRET is 0. In step 2, the ternary complex interacts with mRNA; this gives 0.35 FRET, representing the codon-anticodon recognition event. In step 3, the ternary complex moves the aa-tRNA closer to the P site (0.5 FRET) and is ready for GTP hydrolysis. In step 4, GTP is hydrolyzed by the interaction of EF-Tu with the GAC. In step 5, inorganic phosphate is released and EF-Tu has changed to the GDP-bound form. In step 6, the 3′ end of the aa-tRNA has accommodated at the peptidyl transferase center and FRET increases to 0.75. In step 7, peptide bond formation occurs. Blanchard et al. also analyzed what happens after proper accommodation of the A site tRNA (Blanchard et al., 2004Blanchard 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). Interestingly, they observed FRET fluctuations between two values, 0.74 and 0.45 (Figure 5B; note the time scale change from Figure 5A). The 0.74 FRET peak was attributed to the "classical state" with both the anticodons and the 3′ ends of tRNAs in the A and P sites. The 0.45 FRET state was assigned to the "hybrid state" where the anticodons stay in the A and P sites on 30S but the 3′ ends move to the P and E (exit) sites. After accommodation at the A site, fluctuation between classical and hybrid states occurred with roughly similar populations in each state. However, the lifetime of the classical state was reduced 6-fold by peptide bond formation which therefore increases the probability of the ribosome being in the hybrid state and readying the ribosome for the next step of ribosome translocation. The ribosome is one of the most complex systems studied so far by single-molecule spectroscopy, and there is great potential to look at even more details by combining new structural information and biochemical approaches. For example, the ribosome itself can be labeled by inserting loops in the ribosomal RNA for hybridization of a fluorescently labeled oligomer (Dorywalska et al., 2005Dorywalska M. Blanchard S.C. Gonzalez R.L. Kim H.D. Chu S. Puglisi J.D. Site-specific labeling of the ribosome for singl
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