Single Ribosome Dynamics and the Mechanism of Translation€¦ · 55 Å 50 Å E A GTPaseGTPase...

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Single Ribosome Dynamicsand the Mechanismof TranslationColin Echeverrıa Aitken,1 Alexey Petrov,2and Joseph D. Puglisi2,3

1Biophysics Program, 2Department of Structural Biology, and 3Stanford MagneticResonance Laboratory, Stanford University, Stanford, California 94305-5126;email: [email protected], [email protected], [email protected]

Annu. Rev. Biophys. 2010. 39:491–513

The Annual Review of Biophysics is online atbiophys.annualreviews.org

This article’s doi:10.1146/annurev.biophys.093008.131427

Copyright c! 2010 by Annual Reviews.All rights reserved

1936-122X/10/0609-0491$20.00

Key Wordssingle-molecule, FRET, force, ratcheting

AbstractOur current understanding of the mechanism of translation is based onnearly fifty years of biochemical and biophysical studies. This mecha-nism, which requires the ribosome to manipulate tRNA and step repeti-tively along the mRNA, implies movement. High-resolution structuresof the ribosome and its ligands have recently described translation inatomic detail, capturing the endpoints of large-scale rearrangements ofthe ribosome. Direct observation of the dynamic events that underliethe mechanism of translation is challenged by ensemble averaging inbulk solutions. Single-molecule methods, which eliminate these aver-aging effects, have emerged as powerful tools to probe the mechanism oftranslation. Single-molecule fluorescence experiments have describedthe dynamic motion of the ribosome and tRNA. Single-molecule forcemeasurements have directly probed the forces stabilizing ribosomalcomplexes. Recent developments have allowed real-time observationof ribosome movement and dynamics during translation. This reviewcovers the contributions of single-molecule studies to our understand-ing of the dynamic nature of translation.

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PIC: preinitiationcomplex

ContentsINTRODUCTION . . . . . . . . . . . . . . . . . . 492SINGLE-MOLECULE METHODS

AND TRANSLATION . . . . . . . . . . . . 494Fluorescence Methods . . . . . . . . . . . . . 494Force Methods . . . . . . . . . . . . . . . . . . . . 495

INITIATION . . . . . . . . . . . . . . . . . . . . . . . . 498IF2 and Ribosome Dynamics . . . . . . . 498

THE TRANSITIONTO ELONGATION . . . . . . . . . . . . . . 500

ELONGATION . . . . . . . . . . . . . . . . . . . . . 501tRNA Dynamics on the Ribosome . . 501Ribosome Dynamics . . . . . . . . . . . . . . . 502

TERMINATION . . . . . . . . . . . . . . . . . . . . 504RIBOSOME DYNAMICS

DURING TRANSLATION . . . . . . . 505CONCLUSION . . . . . . . . . . . . . . . . . . . . . 506

A Dynamic Model of Translation . . . 507Future Perspectives . . . . . . . . . . . . . . . . 508

INTRODUCTIONThe translation of genetic information intoproteins consumes nearly half the dry weight ofa dividing bacterial cell and up to 60% of its en-ergy (44). Translation is performed by the ribo-some, a massive molecular machine composedof a large (50S, in bacteria) and a small (30S)subunit (62, 79); both subunits consist of highlystructured RNA and protein elements. The ri-bosome selects aminoacylated tRNA as speci-fied by the mRNA and catalyzes the formationof peptide bonds. Protein factors guide the pro-gression of important mechanistic events dur-ing translation. Translation by the ribosome israpid and precise; the ribosome adds up to 20amino acids to the growing polypeptide chaineach second and commits an error only every1000 to10,000 additions (84). The basic mech-anism and machinery of translation are con-served through all organisms.

Our current understanding of the mech-anism of translation is based on nearly fiftyyears of biochemical and biophysical studies.One of the main challenges in applying bulk

biochemical and biophysical techniques totranslation is the complexity of the process.Translation involves the dynamic interplayof multiple ligands—mRNA, tRNAs, andfactors—through repetitive cycles of peptidebond formation; many of these ligands interactonly transiently with the ribosome. Ribosomecomplexes prepared in vitro rapidly be-come heterogeneous. Early studies employedribosome-targeting antibiotics, nonhydrolyz-able nucleotide analogs, and functional mutantsto stall translation at specific stages. Variouschemically modified tRNA analogs allowedformation of ribosome complexes mimickingfunctional intermediates during elongation.The reconstitution of translation in vitro pro-vided researchers with real-time control overthe complexity and composition of translationexperiments.

These systems made possible the applicationof general biochemical and biophysical tech-niques to the study of translation. Chemicalfootprinting of stalled translational complexesmapped ribosomal active sites and provided ev-idence for the role of conformational dynamicsin translation (49–52). Toeprinting was used tofollow the position of ribosomes on the mRNA,probing translocation and ribosome movementon mRNAs (24, 25). Fluorescent and isotopiclabeling of RNA ligands and protein factors en-abled stop-flow and quench-flow techniques tobe applied to in vitro reconstituted translationsystems (61, 63); these approaches revealed thebasic kinetic mechanisms of the general phasesof translation: initiation, elongation, and termi-nation (38, 40, 64, 65).

Whereas eukaryotes employ a far more so-phisticated regulatory network to tune trans-lation, particularly during initiation, the basalmechanism of translation is similar in bothprokaryotes and eukaryotes. Here we focus onthe simple core mechanism of bacterial transla-tion. During initiation, the ribosome is assem-bled at the start (AUG) codon of the mRNA(41, 42). First, the 30S subunit, mRNA, andinitiator tRNA (fMet-tRNAfMet) assemble toform the 30S preinitiation complex (PIC). Join-ing of the 50S subunit and 30S PIC to form

492 Aitken · Petrov · Puglisi


an elongation-competent ribosome (70S) com-pletes the initiation phase. Initiation is mod-ulated by conserved protein initiation factors(IF1, IF2, and IF3). The ribosome then en-ters the elongation cycle, during which it firstselects an aminoacyl-tRNA molecule comple-mentary to the mRNA codon in its aminoacyl(A) site (60, 78). The ribosome employs thisselected tRNA as a substrate for peptide bondformation, transferring the growing polypep-tide chain from the peptidyl-tRNA in its pep-tidyl (P) site, to the A-site tRNA. Peptide bondformation is followed by translocation, duringwhich the ribosome steps onto the next codonof the mRNA and transfers the A- and P-sitetRNAs into the P and E (exit) sites, prepar-ing the ribosome for another round of elonga-tion. The ribosome is aided during elongationby the protein elongation factors (EF-Tu andEF-G). Once it encounters a stop codon (UAA,UAG, or UGA), the ribosome exits the elonga-tion cycle and begins termination (40, 82). Dur-ing termination, the completed peptide chain isreleased and the ribosomal subunits dissociate.

As with the other phases of translation, termi-nation and recycling of the translational appa-ratus are guided by protein factors, notably therelease factors (RF1 or RF2, and RF3) and theribosomal recycling factor (RRF). Translationrequires external sources of energy to ensurefidelity and processivity, including ATP to syn-thesize aminoacyl-tRNAs and GTP coupled tothe GTPase factors IF2, EF-G, and RF3.

The mechanism of translation, in which theribosome steps repetitively along the mRNA,requires dynamic movement. The role of struc-tural dynamics in translation was suggestedmore than forty years ago (14, 70) and was sub-sequently supported by chemical footprinting(51) and neutron scattering results (71). Therecent high-resolution structures of the ribo-some and its ligands have underscored the roleof large-scale dynamic events during transla-tion. The ribosome has a molecular weight ofnearly 3 MDa, a diameter of roughly 210 to250 A, and ligand binding sites separated byat least 40 A (Figure 1) (15, 83). This scale al-lows the ribosome to bind and manipulate large,

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Figure 1An architectural view of the ribosome. (a) Frontal view of the 70S ribosome. The large subunit (50S) is shown in purple and the smallsubunit (30S) is shown in gray; the mRNA is shown in red. The position of the tRNA binding sites (A, aminoacyl; P, peptidyl; E, exit),the GTPase center, and the L1 stalks are indicated with yellow overlays. The overall dimensions of the ribosome (200–210 A) anddistances between tRNA binding sites (50–55 A) are designated by arrows. (b) E-site lateral view of the 70S ribosome, showing thetunnel occupied by mRNA and tRNA. (c) Relative positions of the tRNAs, in the classical configuration, on the ribosome. Distancesbetween codons on the mRNA (12 A), tRNA elbow regions (50–55 A), and tRNA anticodon and acceptor stems (85 A) are designatedby arrows.

www.annualreviews.org • Ribosome Dynamics and Translation 493


TIRF-M: totalinternal reflectionfluorescencemicroscopy

flexible ligands: !20-kDa tRNAs and proteinfactors up to !100 kDa in size. Structural stud-ies on trapped functional intermediates of thetranslation cycle have linked conformationalrearrangements of the ribosome and ligandsto mechanistic models of translation. Cryo-electron microscopy (cryo-EM) structures ofthe ribosome bound to the GDPNP and GDPforms of IF2 suggest significant rearrangementsof both the ribosome and IF2 as a function ofGTP hydrolysis (4, 59). These structures im-ply a counterclockwise rotation (!6") of the30S subunit, with respect to the 50S subunit.Prior structural studies by Frank and coworkers(2, 23, 80) had suggested that GTP hydrolysisby EF-G may drive similar rotation of the 30Ssubunit during translocation; thus, intersubunitmovement, as in many allosteric proteins, maybe a central feature of ribosome function. Otherstudies, using X-ray crystallography (66, 87),cryo-EM (3, 23, 27, 36), fluorescence (34), andNMR spectroscopy (13, 22, 81), have impli-cated the dynamic movement of the 30S headand 50S L1 and L7/L12 stalks during transla-tion, as well as myriad local rearrangements thatoccur in response to tRNA and factor binding.

These studies provide powerful evidence forthe role of large-scale dynamic motions duringtranslation. Yet current high-resolution struc-tural data yield static information that onlysuggests dynamics. Direct observation of theseevents is challenged by the heterogeneity andcomplexity of translation. Bulk biochemical andbiophysical methods are hindered by tempo-ral and ensemble averaging, especially throughmultiple rounds of translation. Single-moleculemethods eliminate these effects, thus permit-ting the observation of dynamic, transient, andrare events (31, 53, 55). The observation ofsingle ribosomes over the past eight years hassuccessfully revealed the nature of many ofthe dynamic events underlying the translationmechanism (10, 46, 58). Earlier single-moleculetranslation studies focused on the role of tRNAfluctuations during the first round of elongation(11, 12). Recent studies have applied single-molecule methods to the observation of bothglobal and local ribosome dynamics throughout

the three phases of translation. Here, we reviewthe contribution of single-molecule methods toour understanding of key mechanistic eventsduring translation, with a focus on ribosomedynamics.

SINGLE-MOLECULE METHODSAND TRANSLATIONSingle-molecule techniques have emerged aspowerful biophysical tools for probing com-plex biological processes. The observationof single molecules permits the detection ofsubnanometer-distance changes and the mea-surement of piconewton molecular forces with-out averaging, unlike bulk solution techniques(53, 55). Traditional biophysical methods haveelucidated the role of dynamics in simple bio-logical systems but are challenged by the struc-tural and compositional complexity of transla-tion. Fast spectroscopic methods provide hightime resolution (femtosecond to picosecondtimescales) but are not truly needed to ex-plore the relatively slow process of transla-tion. NMR spectroscopy can map dynamicsover a range of timescales in atomic detailbut is hindered by size limitations in solution.The single-molecule approaches described be-low have only limited time resolution (millisec-ond) but are optimally deployed against com-plex, heterogeneous and repetitive processessuch as translation.

Fluorescence MethodsSingle-molecule fluorescence methods providetime-resolved information on the identity andconformation of molecular complexes. Thesetechniques can be broadly divided into two cat-egories: diffusion-based techniques such as flu-orescence correlation spectroscopy (FCS) andimmobilization-based techniques such as totalinternal reflection fluorescence microscopy(TIRF-M) (35, 37). Because TIRF-M providesobservation times on the minute timescaleand with millisecond time resolution, it is thetool of choice in single-molecule fluorescencestudies of translation, which is relatively slow,



taking !100 ms per amino acid in vitro.Immobilization of biomolecules via bi-otin:streptavidin interactions onto polyethy-lene glycol (PEG)-derivatized surfaces allowsfor the discrimination and extended obser-vation of individual molecules. Fluorescentlylabeled molecules can then be delivered usinga syringe pump and illuminated with incidentlaser light. TIRF-M illumination createsan evanescent wave that penetrates into thesurface/solution interface, creating a smallillumination volume to reduce backgroundsignal; fluorescent emission is detected by acharge-coupled device (CCD) chip. Freelydiffusing fluorescent molecules are thus in-visible to detection at concentrations lowerthan 50 nM and are observed only uponbinding to an immobilized biomolecule. Whendifferent biomolecules are labeled with distinctfluorophores, multiple components can beobserved simultaneously. Colocalization offluorescence from distinctly labeled singleligands reports on their simultaneous inter-action with an immobilized molecule. Thesefluorescence tracking experiments can revealthe specific order of molecular events, which isdifficult information to obtain by conventionalbiophysical or biochemical techniques. Thelifetimes of bound (fluorescent) and unbound(nonfluorescent) states can be accumulatedand fit to statistical distributions to describethe kinetic behavior of intermediates invisibleto bulk methods. Analysis of fluorescenceintensities from discrete states can similarlyidentify reaction intermediates (67).

Single-molecule fluorescence resonanceenergy transfer (FRET) occurs throughdistance-dependent dipolar interaction of twofluorophores and reports on dynamic confor-mational changes. The sensitivity of FRETdepends on the fluorophores chosen; the mostpopular FRET pair, the cyanine dyes Cy3 andCy5, provides a signal sensitive to angstrom-scale distance changes over a range of !20 to 80A, following a 1/R6 distance dependency for in-terdye distance (10). Fitting of single-moleculeFRET efficiency distributions can identifyunique conformational states; kinetic data

FRET:fluorescence/Forsterresonance energytransfer

are extracted by statistical analysis of distinctFRET state lifetime distributions (67). High-resolution structures guide specific placementof FRET probes at distinct sites on both the ri-bosome and its ligands. Single-molecule FRETstudies of translation have resolved the processof tRNA selection to angstrom and millisecondresolutions (Figure 2). These methods havealso begun to describe the various local andglobal dynamics of the ribosome and their rolein the mechanism of translation.

Force MethodsThe forces that stabilize molecular complexesand generate their movement can be probedby direct manipulation of single molecules withoptical tweezers. State-of-the-art implementa-tions provide subnanometer and piconewtoninformation on a millisecond timescale (31).Force methods generally require manipulationof single-molecules through two points of at-tachment; these molecular handles can then ex-ert a force on the molecule. The attachmentof micron-sized polystyrene beads provides themeans to manipulate individual biomolecules.Trapping these beads, via radiative pressurefrom a focused infrared laser, allows spatial ma-nipulation and detection (55). Secondary at-tachment of trapped molecules can be achievedby surface immobilization, by using methodssimilar to those used in immobilization-basedfluorescence techniques. Precise movement ofthe surface using a piezoelectric stage displacesa trapped bead from the center of the opticaltrap, thus applying a returning force on the beadand the attached molecular complex. The pre-cise measurement of bead displacement fromthe trap center allows calculation of the ap-plied force, as the optical trap behaves like a lin-ear spring. The exerted force can be increasedgradually until it exceeds the stability of thecomplex; this rupture force reports directly onthe strength of molecular interactions, such asthose between the ribosome and the mRNA(73). Alternatively, molecules are attached totwo beads. The first bead is again held in anoptical trap; the second bead is either held with



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Figure 2Local domain and tRNA dynamics on the ribosome. Fluorescent labeling with the cyanine dyes Cy3 ( green) and Cy5 (red ) establishesfluorescence resonance energy transfer (FRET) signals that report on L1 and tRNA dynamics on the ribosome. FRET between L9 andL1 (panel a, top) and L33 and L1 ( panel b, top), separated by !55 A, reports on the position of the L1 stalk relative to the 50S subunit.The L9:L1 FRET signal reveals fluctuation between open and closed conformational states of the L1 stalk upon peptide bondformation ( panel a, bottom). L1:tRNA (!60 A) and tRNA:tRNA (!50 A) FRET report on the dynamic path of tRNA through theribosome, and on the interaction between the L1 stalk and tRNA ( panel b, top). Similar to L1, tRNAs experience fluctuations uponpeptide bond formation ( panel b, bottom).

a micropipette or captured in a second opticaltrap. This trap design can facilitate bead ma-nipulation or improve the precision with whichsmall distance changes between the two attach-ment points can be measured (54, 69), allowingreal-time observation of molecular movement,such as the translocation of the ribosome alongthe mRNA (Figure 3) (77).

The possible heterogeneity of transla-tional complexes complicates single-moleculeforce studies of the ribosome. Fluorescence

methods allow characterization of molecu-lar complex composition. The application ofsingle-molecule force methods in the study oftranslation has benefited from the incorpora-tion of fluorescence-based sorting of transla-tional complexes, which confirms the integrityof molecular complexes prior to measurement(73).

Single-molecule fluorescence and forcemethods provide dynamic distance informa-tion on the translation apparatus at the


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Figure 3Force methods and translation. (a) Surface immobilization and optical trapping of ribosome complexes allows direct measurement ofthe forces holding the ribosome on the mRNA. Surface-immobilized ribosome complexes are manipulated by trapping an attachedpolystyrene bead with a focused infrared laser beam. Displacement of the bead from the center of the optical trap applies a constantlyincreasing force to the ribosome complex, which eventually ruptures, allowing the bead to return to the trap center (top). The positionof the bead at the rupture event allows calculation of the stability of the ribosome:mRNA interaction (bottom). (b) Precise measurementof mRNA length reports on the ribosome’s position on the mRNA. An mRNA hairpin is stretched between two polystyrene beads, oneof which is held at constant force by an optical trap. Translation by the ribosome forces hairpin melting, allowing extension of themRNA (top). mRNA extension, and thus ribosome position, can be followed in real-time at subnanometer precision, revealingindividual translocation events (bottom).


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SD: Shine-Dalgarno

IC: initiation complex

Hybrid state:staggered tRNAconfiguration in whichthe acceptor stems ofA- and P-site tRNAoccupy the adjacentsite P and E sites, orA/P and P/E

PTC: peptidyl-transferase center

subnanometer scale, and with the millisecondresolution relevant to the events of translation.These techniques have provided fascinating in-sights into the dynamic sequence of events un-derlying all three phases of the translation cycle.

INITIATIONInitiation is the coordinated assembly of 50Sand 30S ribosomal subunits, initiator tRNA,mRNA to form an elongation-competent ri-bosome at the start codon of the mRNA (41,42, 45). Initiation prepares the ribosome to en-ter the energy-expensive elongation cycle andis highly regulated to avoid improper levels ofgene expression or accumulation of aberrantproteins due to out-of-frame translation. Ini-tiation begins with positioning of the 30S sub-unit and tRNA at the start codon of the mRNAto form the 30S PIC. This complex is stabi-lized by base pairing between the mRNA Shine-Dalgarno (SD) sequence and the 30S anti-SDsequence, as well as between the AUG codonand the anticodon of the initiator tRNA. Initi-ation factors IF1 and IF3 guide assembly of the30S PIC by positioning the mRNA and initiatortRNA in the P site of the 30S subunit and pre-venting premature association of the 50S sub-unit and A-site tRNA (6, 7, 19, 32). IF2 recog-nizes the formyl group of fMet-tRNAfMet andstabilizes its interaction with the 30S subunit(5, 33). IF2 also plays a central role in the de-cisive event of translation initiation, joining ofthe 50S subunit and 30S PIC to form the 70Sinitiation complex (IC). IF2 accelerates subunitjoining, which triggers a cascade of events thatultimately prepare the ribosome to enter theelongation cycle: hydrolysis of GTP by IF2,dissociation of the initiation factors, and ar-rival of the first elongator tRNA. Eukaryotictranslation initiation is a more complex pro-cess, requiring at least 11 protein factors andgoverning multiple regulatory pathways (38).It is distinguished by a scanning step, in whichpreinitiation complexes assembled at the 5# endof the mRNA locate the start codon throughan ATP-dependent process hypothesized to

involve movement of the preinitiation complexalong the mRNA.

Recent structural studies have illuminatedprokaryotic the 30S PIC (68) and 70S IC (4, 59)in atomic detail. Cryo-EM structures of the 30SPIC show fMet-tRNAfMet in the 30S P site ina rotated conformation similar to that of tRNAin the hybrid (P/E) state. IF2 spans the inter-subunit face of the 30S subunit while contact-ing both the acceptor (-CCA) end of initiatortRNA and regions of the 30S subunit respon-sible for forming intersubunit bridges with the50S subunit. Cryo-EM structures of the 70SIC trapped (with the nonhydrolyzable analogGDPNP) prior to GTP hydrolysis show thedomain II/III of IF2 contacting the GTPase-activating center of the 50S subunit. Structuresof this complex after GTP hydrolysis reveal arotation of the 30S subunit and accommodationof the 3# end of initiator tRNA in the peptidyl-transferase center (PTC) of the 50S subunit.Remarkably, this rotation of the 30S subunit re-sembles that previously observed in structuresof the ribosome before and after GTP hydrol-ysis by EF-G (1, 36, 75).

IF2 and Ribosome DynamicsRecently, single-molecule FRET methods wereapplied to probe the dynamic events of trans-lation initiation. Marshall et al. (47) followedFRET between the two ribosomal subunitsduring initiation by employing a labelingstrategy developed previously (Figure 4b).Dorywalska et al. (18) engineered metastableRNA sequences into surface-accessible, phylo-genetically variable hairpin loops in both the30S and 50S subunits using mutagenesis. Hy-bridization of complementary and fluorescentlylabeled DNA oligos permits specific and effi-cient labeling of the two ribosomal subunitswithout disturbing function. Labeling with Cy3at 30S helix 44 and with Cy5 at 50S helix 101—separated by !45 A according to available struc-tural data—provides a FRET signal that reportson the global orientation of the two subunits yetis distant (>120 A) from the active sites and dy-namic regions of the ribosome.



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Figure 4Global ribosome dynamics. Fluorescent labeling with the cyanine dues Cy3 ( green) and Cy5 (red ) establishesa fluorescence resonance energy transfer (FRET) signal that reports on intersubunit dynamics. (a) Steady-state observation of ribosomes labeled at L9 and S6 or S11. FRET reveals spontaneous conformationalfluctuations similar to those observed for the L1 stalk and tRNA (bottom). (b) FRET between H44 (30S) andH101 (50S) reports on the relative orientation of the two ribosomal subunits. Real-time delivery experimentsreveal irreversible transitions between the nonrotated and rotated states of the ribosome, driven by peptidebond formation and the action of EF-G (bottom).

Surface immobilization of Cy3-30S PICs,followed by delivery of Cy5-50S subunits un-der Cy3 excitation at 532 nm, provides a robustsingle-molecule FRET assay for initiation. 50Sjoining to immobilized 30S PICs results in anintersubunit FRET interaction that is sensitiveto small (5 A) changes in the relative orienta-tion of the two ribosomal subunits. This systemrecapitulates many of the previously describedeffects of divalent Mg2+ ions and polyamineson subunit joining during initiation, underscor-ing the role of electrostatic effects in this pro-cess. IF2 binding to the 30S PIC accelerates 50Ssubunit joining more than fourfold; addition of

either IF1 or IF3 slows the process, consistentwith these factors preventing premature 70Sassembly.

These single-molecule measurementsdemonstrated that IF2 guides proper assemblyof the 70S IC upon subunit joining. In theabsence of initiation factors, ribosomes assem-ble into two distinct conformations; the meanFRET intensities of these conformations areconsistent with cryo-EM structures pre- andpost-GTP hydrolysis by IF2. The addition ofthe initiation factors enriches the elongation-competent, high-FRET conformation. Themean FRET value (!0.45) for this state is


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consistent with cryo-EM structures of theribosome after GTP hydrolysis by IF2. Inthe presence of the nonhydrolyzable analogGDPNP, ribosomes initiate exclusively in thelow-FRET conformation, which is unable toenter elongation; the mean FRET value for thisstate (!0.35) is consistent with structures ofthe ribosome prior to GTP hydrolysis by IF2.These results suggest a model in which initial50S joining yields a 70S complex in whichthe 30S subunit is rotated; GTP hydrolysisby IF2 might then drive rotation of the 30Ssubunit into the aligned state and committhe ribosome to the elongation cycle. Directobservation of subunit joining at fast (20 ms)data acquisition rates revealed this sequentialassembly mechanism. Ribosomes initiallyassemble in the low-FRET conformation,followed quickly by a transition to the high-FRET, elongation-competent conformation.The lifetime of the transient low-FRET state(!30 ms) agrees with experimentally deter-mined rates for GTP hydrolysis by IF2 (64).Subunit rotation is likely coupled to movementof the 3# end of initiator tRNAfMet from anE-site location near L1 to the canonical P sitein the PTC, where it can act as a substrate forpeptidyl transfer. 30S rotation, which requiresan external energy source, may govern thesemovements and serve as a checkpoint beforeentry into the elongation cycle.

THE TRANSITIONTO ELONGATIONThe first round of elongation, in which elon-gator tRNA arrives at a ribosome primed onlywith initiator tRNA in the P site, is unique.The ribosome, which at this point remains as-sociated with the SD sequence of the mRNA,must synthesize the first peptide bond and takethe first of many codon steps along the mRNA.How translation balances the need for stablemRNA:ribosome interaction during initiationand elongation with the requirement for move-ment remains a key unanswered question.

Uemura et al. (73) employed optical tweezermethods to measure the force with which single

ribosomes grip the mRNA during the transitionfrom initiation to elongation (Figure 3a). Theforce required to rupture the mRNA:ribosomeinteraction was determined for distinct 70SICs assembled on mRNAs containing eithera strong natural SD sequence or no SD se-quence. 70S complexes containing deacylatedP-site tRNAfMet were stabilized by !5 pN,compared with complexes assembled withouttRNAs regardless of SD sequence identity. Theaddition of A-site Phe-tRNAPhe further stabi-lized these complexes by another !10 pN. Forboth complexes, the mRNA:ribosome interac-tion was approximately 10 pN greater on themRNA containing a strong SD sequence, high-lighting the role that the SD interaction playsin stabilizing the 70S IC. Complexes assem-bled with N-acetyl-Phe-tRNAPhe—a peptidyl-tRNA analogue—in the A site were destabilized(up to 13 pN) on both mRNAs. This findingis consistent with prior studies demonstratingthe ability of the ribosome to sense the chem-ical identity of tRNAs bound in the P and Asites. The stability of these complexes was notaffected by the SD sequence, suggesting thatsynthesis of the first peptide bond disrupts theSD interaction. Similar results were obtainedwith 70S complexes first assembled with Phe-tRNAPhe and subsequently permitted to cat-alyze peptide bond formation.

These results underscore the physical re-quirements of ribosome translocation. At eachcodon, the ribosome must first establish andpreserve its reading frame during tRNA selec-tion and peptide bond formation. This likelyinvolves stabilization of the mRNA:ribosomeinteraction. Reading-frame maintenance isextremely important during initiation and thetransition to elongation, a fact highlightedby the stability of the SD interaction. Uponpeptide bond formation, the ribosome mustthen translocate onto the ensuing codon, whichmay be facilitated by a weakening of themRNA:ribosome interaction. The manner inwhich the ribosome modulates its interactionwith the mRNA in response to the events oftRNA selection and peptide bond formation re-mains unclear.



ELONGATIONDuring elongation, the ribosome repetitivelyselects aminoacyl-tRNA, adding the aminoacids they carry to the growing peptide chainvia peptide bond formation. Aminoacyl-tRNAsare selected at the A site of the ribosome ina multistep mechanism facilitated by EF-Tu,a ribosome-activated GTPase (65, 84). EF-Tu,aminoacyl-tRNA, and GTP assemble to forma ternary complex that enters the ribosomevia protein:protein interactions between EF-Tuand the tetrameric 50S protein L7/L12. Recog-nition of cognate tRNA by pairing between themRNA codon and tRNA anticodon stem in the30S A site enhances the GTPase activity of EF-Tu. GTP hydrolysis and dissociation of EF-Tu permit full accommodation of aminoacyl-tRNA, positioning its 3# end in the PTC of the50S subunit. The ribosome then catalyzes theformation of a peptide bond, transferring thenascent chain onto the A-site tRNA and leav-ing a deacylated tRNA in the P site. Followingpeptide bond formation, the ribosome translo-cates the A- and P-site tRNAs into the P andE sites, stepping onto the next codon of themRNA (78, 79). Although it can occur sponta-neously, albeit slowly, translocation is catalyzedby a second ribosomal GTPase, EF-G (8, 29).Elongation occurs via a similar mechanism ineukaryotes, with eEF1A and eEF2 playing theparts of EF-Tu and EF-G, respectively.

As with initiation, structural evidence impli-cates ribosome dynamics in the mechanism ofelongation. Cryo-EM studies suggest that uponpeptide bond formation, the 30S subunit ro-tates with respect to the 50S subunit (1, 36, 75).In this rotated, or ratcheted conformation, the3# ends of the A- and P-site tRNAs protrudeinto the P and E sites, respectively. This tRNAconfiguration, known as the hybrid state, is apretranslocation intermediate and may be fa-cilitated by 30S rotation (3). Reverse rotationof the 30S subunit may then drive full translo-cation of the tRNAs and step the ribosome ontothe next codon of the mRNA. High-resolutionX-ray structures have revealed new intermedi-ates in this process and implicate a motion of the

Cognate:tRNA:codon matchwith Watson-Crickpairing at allnonwobble positions,and acceptablemismatches at thewobble position

30S head domain during 30S ratcheting (87).The mobile L1 and L7/L12 stalks also likelyassist tRNA movement through the ribosome(1, 9). These local dynamic events may coordi-nate tRNAs as they travel in steps up to 40 Aduring their transit through the ribosome.

tRNA Dynamics on the RibosomeInitial applications of single-molecule meth-ods to the study of translation focused on thenature of tRNA dynamics on the ribosome(11, 12, 43). The elbow region of L-shapedtRNAs were labeled with fluorophores via spe-cific attachment chemistry to hypermodifiednucleotides (Figure 2b). Delivery of ternarycomplex containing Cy5-Phe-tRNAPhe to im-mobilized ribosomes primed with Cy3-fMet-tRNAfMet in the P site established a FRET sig-nal that reports on the path of incoming tRNAthrough the ribosome (11). Postsynchroniza-tion of this FRET signal eliminates temporalaveraging effects from bimolecular arrival ofternary complexes, revealing the progression ofincoming A-site tRNA through three discretestates. These states were assigned with the aidof existing structural information and the use ofribosome-targeting antibiotics as biochemicalcontrols; this analysis assumed that P-site tRNAdoes not move significantly during selection,such that FRET changes reflect A-site tRNAmovement. Initial binding of ternary complexat L7/L12 is invisible to the FRET interaction,as the A-site tRNA is more than 100 A from theP-site tRNA. Upon codon recognition, a tran-sient (!50 ms) low-FRET (!0.35) state is ob-served, followed by a mid-FRET (!0.5) statesignaling successful activation of EF-Tu. Fullaccommodation of tRNA in the A site yields ahigh-FRET (!0.75) state.

The dynamic pathway of tRNA selectionhighlights the role of conformational allosteryduring translation. Upon establishment of lowFRET, A-site tRNAs rapidly and reversiblysample the mRNA codon prior to GTPase ac-tivation. The antibiotic tetracycline stalls selec-tion at this phase, as does thiostrepton, which



SRL: sarcin-ricin loop

Nonrotatedribosome: globalconformation of theribosome prior topeptide bondformation

inhibits the GTPase activity of ribosome-associated GTPases. Both antibiotics block sta-ble progression of the ternary complex to theGTPase activation state by either blockingmovement of the tRNA (tetracycline) at the30S subunit or sterically blocking the GTPasecenter (thiostrepton) on the 50S subunit (11,30). These results suggest that stabilizing con-tacts in the mid-FRET state may serve as an al-losteric signal for GTPase activation. Cleavageof the sarcin-ricin loop (SRL) with the toxinrestrictocin allows tRNA to access the mid-FRET state but slows overall progression to thefully accommodated (high-FRET) state, con-sistent with a model in which SRL stimula-tion of EF-Tu requires stable tRNA position-ing in the GTPase-activated state. Kirromycin,which uncouples GTP hydrolysis from confor-mational changes in EF-Tu, stalls the tRNA inthe mid-FRET state.

Upon arrival at the high-FRET state, theA-site tRNA is fully accommodated within thePTC, poised for rapid peptide bond formation.After peptide bond formation, tRNAs fluctu-ate between the high-FRET state and a sec-ond mid-FRET (!0.45) state (12, 39). This0.45 FRET state was assigned to the hybridstate, in which the 3# ends of the A- and P-sitetRNAs are shifted into the P and E sites of the50S subunit, with a longer distance betweenthe two tRNAs. The hybrid state is thought toprecede full translocation by the ribosome andhas recently been captured in cryo-EM struc-tures of pretranslocation ribosome complexes(1, 3). Deacylated tRNA in the P site favors thehigh-FRET state, or classical tRNA configura-tion. The presence of N-acetyl-Phe-tRNAPhe

encourages hybrid state formation. These re-sults support prior biochemical data suggestingthat peptide bond formation favors formationof the hybrid state, thus preparing the ribosomefor translocation. Recently, Munro et al. (57)were able to discriminate between two distincthybrid states. The first state resembled the pre-viously described hybrid state, with the 3# endsof both tRNAs pointing into the adjacent siteson the 50S subunit. In the second, metastablestate, only the P-site tRNA is directed into the

adjacent 50S site. Increased peptide length en-riched hybrid-state occupancy, suggesting thatelongating ribosomes may drive formation ofthe hybrid state, and thus translocate, with in-creasing efficiency. The detection of this novelstate underscores the ability of single-moleculemethods to observe transient intermediates incomplex systems.

Ribosome DynamicsA major goal of single-molecule translationstudies is to follow directly the dynamic move-ments of the ribosome, including both localdomain and intersubunit motions. How do-main and subunit movements are coupled tothe chemical steps of translation remains un-clear. Structural methods have captured twoglobal states of the ribosome that are coupled todynamic tRNA positioning. In the rotated, orratcheted, ribosome, the A- and P-site tRNAsadopt a hybrid configuration, whereas the non-rotated ribosome stabilizes the classical config-uration of tRNA. Recent single-molecule stud-ies have directly probed ribosome dynamics.

Cryo-EM reconstructions have implicatedlarge-scale motions of the conserved 50S L1stalk, which consists of 23S rRNA and proteinL1, in the mechanism of translocation (1, 36).The L1 stalk moves !20–40 A toward the Esite in response to the action of EF-G. Usinga tRNA:tRNA FRET signal, Munro et al. (57)observed that depletion of the L1 protein stabi-lizes the classical configuration of tRNA on theribosome up to 45%, compared with wild type,suggesting that dynamic movements of the L1stalk may help coordinate tRNA movement.More recently, Fei et al. (21) followed FRETbetween the L1 stalk and tRNA in either E orP sites (Figure 2b). Equilibrium FRET (L1:P-site tRNA) analysis of complexes containingdeacylated P-site tRNA shows fluctuations be-tween two states, which the authors assignedto the hybrid and classical tRNA configura-tions, consistent with previous observations oftRNA dynamics after peptide bond formation.The higher FRET value (!0.8) was assignedto the hybrid state, and is consistent with a



conformation in which the L1 stalk closes toform stable contacts with the E-site tRNA. Thelower FRET value (!0.2) was assigned to theclassical state of tRNA and is consistent with aconformation in which the L1 stalk opens. Theaddition of EF-G ·GDPNP resolves thesefluctuations, stabilizing the high-FRET state.Real-time delivery of EF-G results in stable for-mation of the high-FRET state. These resultsare consistent with a model in which L1 closesto stabilize the hybrid state and subsequentlyescorts the incoming E-site tRNA duringtranslocation. However, L1:tRNA FRET doesnot report on the orientation of the L1 stalkrelative to the ribosome or on global ribosomemovements, such as ratcheting.

Single-molecule FRET between the L1stalk and other regions of the 50S subunit hascorroborated models for L1 motion based oninterpretation of L1:tRNA FRET data. Simi-lar to previous results, Fei et al. (20) observedFRET between L1 and L9 to fluctuate be-tween two levels in pretranslocation complexes(Figure 2a). Again, the high-FRET state wasassigned to the closed conformation of the L1stalk, and the low-FRET state was assignedto the open conformation. Comparison of therates of L1 opening and closing obtained withthe L1:tRNA and L1:L9 FRET signals suggeststhat both signals report on the same process.Nonetheless, comparison of these two FRETsignals upon delivery of EF-G provides newdetail on the role of L1 dynamics. WhereasL1:tRNA FRET proceeds to a stable high-FRET state upon delivery of EF-G and translo-cation, the L1:L9 signal fluctuates betweenthe open and closed states. For authenticallytranslocated tRNA, these fluctuations are bi-ased to the open conformation; artificial de-livery of deacylated tRNA to the E site favorsthe closed conformation of the L1 stalk. Anal-ogous to these results, Cornish et al. (17) ob-served an open L1 stalk conformation in post-translocation complexes. Artificial delivery ofaminoacyl-tRNA to the E site resulted in anintermediate conformation of the L1 stalk, de-scribed as half-open. The sum of these resultssuggests a model in which opening of the L1

Classical state: tRNAconfiguration in whichthe acceptor andanticodon stems of A-and P-site tRNAoccupy the same site,or A/A and P/P

ASL: acceptor stemloop

stalk after translocation facilitates dissociationof the E-site tRNA. The sensitivity of L1 dy-namics to E-site tRNA is consistent with directinteraction between these sites. However, therelationship between L1 dynamics and globalribosome dynamics is unclear.

The promise of real-time observation ofglobal ribosome dynamics during elongationhas driven multiple recent single-moleculetranslation studies. The counterclockwiseratcheting of the 30S subunit observed inX-ray and cryo-EM structures of pretranslo-cation ribosomes suggests that FRET be-tween the two ribosomal subunits might pro-vide a signal for these dynamics. Cornishet al. (16) labeled single-cysteine mutants ofL9, S6, and S11 to allow site-specific label-ing with fluorescent dyes (Figure 4a). Re-constitution of labeled L9, S6, and S11 withribosomes purified from deletion strains ofthese proteins yielded two sets of FRET-labeled ribosomes, 70S:S6(Cy5)/L9(Cy3) and70S:S11(Cy5)/L9(Cy3), that were shown tobe 50–60% and 80–100% active in in vitrotranslocation and translation assays, respec-tively. 70S:S6(Cy5)/L9(Cy3) ribosomes assem-bled with deacylated tRNAfMet in the P siteand N-acetyl-Phe-tRNAPhe in the A site—pretranslocation complexes—showed sponta-neous fluctuations between two FRET levels(!0.6 and !0.4). The authors assigned thehigh-FRET state to the nonrotated confor-mation of the ribosome, in which the tRNAswere assumed to adopt the classical configu-ration. The low-FRET state was assigned tothe rotated, or ratcheted, conformation of theribosome, in which the tRNAs were assumedto adopt the hybrid state. Incubation of thesecomplexes with EF-G ·GTP favored forma-tion of the high-FRET (classical) state, whereasposttranslocation complexes containing deacy-lated tRNAfMet in the P site preferred the low-FRET (hybrid) state. Ribosomes containing ei-ther an acceptor stem loop (ASL) or no tRNAin the P site preferred the low-FRET state,suggesting that interactions between the 3# endof deacylated P-site tRNA and the 50S E sitemay be involved in stabilizing the hybrid state.



Near-cognate:tRNA:codon pairingwith one mismatch atone of the nonwobblepositions

The low-FRET, hybrid state is kinetically fa-vored in posttranslocation complexes; deacy-lated tRNAfMet increases high-FRET (classical)$ low-FRET (hybrid) transition rates, con-sistent with previous observations of increasedtRNA dynamics upon peptide bond formation.Wang et al. (76) also observed dynamic fluc-tuations between EF-G and L11 upon pep-tide bond formation that were interpreted tobe a result of ribosome unlocking prior totranslocation.

Marshall et al. (48) followed intersubunitFRET to explore the global dynamics of theribosome during the first round of elongation.Cy3-30S and Cy5-50S were labeled at H44and H101 as described above (Figure 4b). Pre-assembled Cy3-30S PICs were immobilized viabiotinylated mRNA in a background of sat-urating fMet-tRNAfMet and IF2, followed bydelivery of Cy5-50S subunits. In this experi-mental design, subunit joining during initia-tion is observed in real time and can serve asa control to eliminate nonfunctional ribosomecomplexes. 50S joining to form an elongation-competent 70S complex establishes FRET be-tween the two subunits and provides a true real-time signal for the relative orientation of thetwo subunits.

Initiation results in the formationelongation-competent 70S complexes inthe high-FRET (0.45) state. If elongation isallowed to occur, by inclusion of the Phe-tRNAPhe · EF-Tu ·GTP ternary complex,FRET trajectories show the high-FRET,nonrotated ribosome state, quickly followedby a single transition to a low-FRET (!0.35)state. This FRET efficiency is consistent withthe rotated, or ratcheted, state of the ribosome.Spontaneous transitions between these twostates are not observed on the timescale(minutes) of these measurements, indicatingthat they are separated by a significant energybarrier. 30S ratcheting requires the reorderingof the RNA:RNA intersubunit bridges, whichlikely requires an external energy source. Whenperformed with Cy2-labeled Phe-tRNAPhe,this experiment permits the correlation ofribosome ratcheting with tRNA arrival at

the ribosome. This three-color experimentreveals that 30S rotation occurs concomitantlywith tRNA arrival. However, ratcheting is notobserved with delivery of Phe-tRNAPhe to70S complexes assembled with a near-cognatecodon (CUU) in the A site. The peptidyl-transferase inhibitor chloramphenicol slowsthe high-low FRET transition up to 50%,suggesting that peptide bond formation, andnot actual tRNA binding or GTP hydrolysisby EF-Tu, is the underlying reaction coupledto ratcheting. Confirming this hypothesis,delivery of puromycin, in the absence of theternary complex, also drives the ratchetingtransition.

These single molecule results suggestthat the energy of peptide bond formation(!%8 kcal mol%1) may provide the impetusfor 30S ratcheting. Delivery of EF-G reversesratcheting, returning 70S complexes to thehigh-FRET, nonrotated state. Inhibition of EF-G dissociation with fusidic acid has no effecton this reverse rotation, suggesting that EF-G-catalyzed GTP hydrolysis (!%10 kcal mol%1)may drive reversion to the nonrotated state dur-ing translocation. These results provide directevidence of the role of global dynamics dur-ing the elongation cycle. Intersubunit dynam-ics are tightly controlled by the stabilizing net-work of RNA:RNA contacts bridging the 30Sand 50S subunits. 30S rotation occurs only inresponse to the irreversible chemical events—peptide bond formation and GTP hydrolysis—that define the elongation cycle, thus provid-ing a physical means of controlling ribosomefunction.

TERMINATIONUpon encountering a stop codon in the A site,the ribosome begins the process of termination,which releases the newly synthesized nascentchain and disassembles the elongating riboso-mal complex, recycling its components for newrounds of translation (40). The stop codon isrecognized by one of two class I release factors,RF1 or RF2, followed by release of the pep-tide chain, leaving a deacylated tRNA in the



P site. Dissociation of RF1/2 from this riboso-mal release complex (RC) is driven by yet an-other ribosomal GTPase, RF3 (26, 85). Struc-tural evidence suggests that GTP hydrolysis byRF3 promotes conformational changes in theribosome similar to those observed upon pep-tide bond formation (28). Specifically, struc-tures of the RC bound to RF3 ·GDP showthe 30S subunit rotated with respect to the 50Ssubunit. After GTP hydrolysis by RF3, ribo-some recycling factor (RRF), in collaborationwith EF-G and IF3, forces the dissociation ofthe RC, thus freeing the ribosomal subunits,tRNA, and mRNA (86). In eukaryotes, the re-lease factor eRF1 is responsible for peptide re-lease from all stop codons. The role of eRF3,a GTPase analogous to RF3, is unknown, andthe mechanism of ribosome recycling remainsuncharacterized (38).

Sternberg et al. (72) used L1:tRNA andRF1:tRNA FRET signals to describe the dy-namic events of translation termination inprokaryotes. FRET between RF1 and P-sitefMet-Phe-(Cy3)tRNAPhe shows that the releasefactor stably binds in the A site, establishing asingle high-FRET distribution (!0.95). ThisFRET efficiency is consistent with X-ray struc-tures of RF1 bound to the ribosome. The sta-bility of the RF1:tRNA FRET signal suggeststhat deacylation of P-site tRNA by RF1 doesnot induce tRNA dynamics like those that oc-cur upon deacylation by incoming tRNA dur-ing elongation. This result is confirmed by theL1:tRNA FRET signal, which also shows nodynamics in the presence of A-site RF1; stablelow FRET (!0.2) is observed before and af-ter delivery of RF1 to ribosomes with peptidyl-tRNA in the P site and an empty A site. RF1thus appears to restrict local dynamics of theL1 stalk and tRNA on the ribosome. Domains2–4 of RF1, which occupy the A site muchlike A-site tRNA in the classic configuration,are sufficient to restrict local ribosome dynam-ics. Binding of RF3 ·GTP and subsequentGTP hydrolysis force the dissociation of RF1and stabilize the ribosome in the high-FRET(!0.8) state. The authors suggest a mech-anism whereby RF3 ·GDP recognizes the

RC: release complex

RF1-bound conformation of the ribosome (L1open and classic P-site tRNA) and exchangesGDP for GTP, thus driving conformationalchanges that force the dissociation of RF1 andthe formation of a closed-L1/hybrid tRNA con-figuration prior to GTP hydrolysis. The effectof RRF on this conformational equilibrium ismore subtle; binding of RRF competes withthese dynamics and, at high concentrations, ap-pears to speed the formation of the closed-L1/hybrid tRNA conformation. Perhaps mostinteresting, however, is the similarity of theconformational cycle observed during termina-tion and elongation. Though subtle differencesexist between the effects of various ligands dur-ing termination and elongation, both processesappear to regulate the dynamic equilibrium be-tween these states.

RIBOSOME DYNAMICSDURING TRANSLATIONThe results discussed thus far come from stud-ies focused on discrete events during transla-tion. Single-molecule fluorescence and forcetechniques have interrogated events during ini-tiation, the first round of elongation, and ter-mination. Yet single-molecule methods permitobservation of dynamic events throughout thetranslation cycle. Recent results highlight thepower of single-molecule methods to probe dy-namic events and detect rare or transient inter-mediate states during all phases of translation.

Sensitive optical trapping techniques havebeen employed to follow the movements ofsingle ribosomes along an mRNA (77). By at-taching micron-sized polystyrene beads to bothends of a single mRNA hairpin, Wen et al. (77)manipulated the mRNA with a micropipetteat the 5# end and an optical trap at the 3#

end. This optical trap exerts a small constantforce sufficient to stretch linear regions of themRNA. As a result, changes in the end-to-enddistance of the mRNA can be measured withsubnanometer resolution. This system permit-ted real-time tracking of the ribosome move-ment along the mRNA, as hairpin melting by atranslating ribosome results in changes to the



Rotated or ratchetedribosome: globalconformation of theribosome in which thesmall subunit is rotatedcounterclockwise withrespect to the largesubunit

overall mRNA length, detected as bead dis-placement toward the center of the optical trap.Ribosome movement was observed as a series ofpauses and codon-sized (2.7 nm, or the meltingof three base pairs) translocation events. Thislandmark study represents the first real-timeobservation of ribosome movement along anmRNA. Moreover, the ability to measure smallchanges in mRNA structure imposed by the ri-bosome makes possible the interrogation of lo-cal mRNA structure effects in processes such asframeshifting.

FRET provides a distinct perspective of ri-bosome movement during multiple rounds oftranslation. To explore the substeps involvedin each round of translation, we expanded onthe intersubunit FRET system established byMarshall et al. (Figure 4b) (C.E. Aitken &J.D. Puglisi, unpublished results, 48). Immo-bilization of Cy3-labeled PICs via biotinylatedmRNA coding for multiple Phe (UUU) codons,followed by real-time observation and deliv-ery of Cy5-50S, Phe-tRNAPhe · EF-Tu ·GTPternary complex, and EF-G ·GTP, yields in-tersubunit FRET trajectories displaying multi-ple high-low-high FRET cycles, akin to the cy-cle previously observed during the first roundof elongation.

Similar experiments using a three-codonmessage coding for Phe(UUU)-Lys(AAA)-Phe(UUU) allow for control over the num-ber of codons read by immobilized ribosomes.Exclusion of Lys-tRNALys ternary complexyields FRET trajectories with one high-low-high FRET cycle. Inclusion of both Phe-tRNAPhe and Lys-tRNALys ternary complexesresults in FRET trajectories containing up tothree FRET cycles. Repetition of this exper-iment with Cy2-labeled Phe-tRNAPhe revealsa burst of blue fluorescence, indicating sta-ble tRNA arrival on the ribosome, concomi-tant with the first and third high-low FRETtransitions. No blue fluorescence was detectedat the second high-low FRET transition, con-sistent with the arrival and incorporation ofunlabeled Lys-tRNALys at the second codonof the mRNA. Counting complete high-low-high FRET cycles on mRNAs of varying length

further confirmed that each FRET cycle resultsfrom ribosome dynamics during one round ofelongation at a distinct codon of the mRNA.This high-low-high FRET cycle represents oneround of elongation from the perspective ofglobal ribosome dynamics.

This system permits the interrogation ofdiscrete molecular events occurring on eachcodon of the mRNA during translation. Fol-lowing intersubunit FRET on translating ri-bosomes probes the component events ofelongation (tRNA selection and translocation)on any mRNA, unlike the force measure-ments described above. Analysis of the life-times of nonrotated and rotated (ratcheted) ri-bosomes at each codon of a 12-codon mRNAhints at the origins of ribosome processivity.Whereas nonrotated state lifetimes show littlecodon dependency, the lifetime of the rotated(ratcheted) state—where the ribosome waits totranslocate—decreases sharply after the firstcodon and continues to decline steadily as theribosome penetrates deeper into the message.Upon its arrival at the sixth codon, the ribo-some has increased its translocation rate morethan twofold. This approach also reveals the de-tailed effects of antibiotics on distinct steps ofthe translation cycle. Real-time observation ofintersubunit dynamics via FRET should permitthe observation of ribosome dynamics from ini-tiation to termination.

CONCLUSIONThe complex mechanism of translation re-quires the ribosome to orchestrate the dy-namic interplay of many ligands. To achievethis mechanistic flexibility, the ribosome un-dergoes large-scale conformational rearrange-ments at key points in translation. In the last tenyears, single-molecule fluorescence and forcestudies have clarified the nature of these dy-namic events. Borrowing from previous bulkstudies, many measurements have focused onthe steady-state behavior of stalled ribosomalintermediates. This was a necessary first stepto confirm the ability of single-molecule sys-tems (both force and FRET) to recapitulate


alexey

Replace "C.E. Aitken & J.D. Puglisi, unpublished results" with reference 14a


translation. More recent efforts have leveragedthe full power of single-molecule methods tofollow translating ribosomes in real time. Theperspective afforded by the observation of sin-gle ribosomes has furthered our understand-ing of core events throughout all phases of thetranslation cycle.

A Dynamic Model of TranslationThe results obtained from single-moleculestudies of translation, when interpreted withinthe context of existing structural and biochem-ical models, demonstrate the central role forglobal ribosome dynamics during translation.The observation of 30S rotation, or ratchet-ing, during both the initiation and elonga-tion phases of translation suggests that thehighly regulated transition between the nonro-tated and rotated (ratcheted) conformations ofthe ribosome may represent a universal mech-anism exploited by distinct ligands to driveevents throughout translation (47, 48). Single-molecule analysis suggests that there is anenergy barrier to relative rotation of the two ri-bosomal subunits, which are stabilized primar-ily by a network of RNA:RNA contacts. Rear-rangement of these contacts likely requires freeenergy provided by the various chemical events,notably GTP hydrolysis and peptide bond for-mation, that delineate the translation cycle.During initiation, the ribosome is assembled inthe rotated state. GTP hydrolysis by IF2, onlyupon proper and complete assembly of the 70SIC, drives 30S rotation and commits the nonro-tated ribosome to the elongation cycle. Duringelongation, selection of cognate tRNA and pep-tide bond formation signals ratcheting of the30S subunit; similar to the role of IF2, EF-G re-verses this rotation, stepping the ribosome ontothe next mRNA codon. Though not yet visual-ized directly, structural studies predict a similarrole for 30S rotation during termination andrecycling (28). How the ribosome transducesthe energy of GTP hydrolysis to drive subunitrotation remains an outstanding question.

In contrast to the regulated ratcheting of the30S subunit, tRNAs experience spontaneous

fluctuations as they pass through the ribosome.tRNA selection occurs through controlled fluc-tuations of the cognate ternary complex along apathway from initial selection, through codon-anticodon pairing, GTPase activation, andeventual accommodation of the aminoacyl-tRNA 3# end in the A site of the PTC (11,43). Once selected at the A site, tRNA areheld firmly in the classical configuration. How-ever, upon peptide bond formation, both A- andP-site tRNA freely fluctuate between classicaland hybrid configurations (12, 39, 57). Thesespontaneous motions are mirrored by thoseof the 50S L1 stalk, suggesting it closes uponpeptidyl transfer, subsequently accompanyingP-site tRNA as it fluctuates (17, 20, 21).Translocation by EF-G opens the L1 stalk andlocks the tRNA in the P and E sites.

The combination of observed spontaneousmotions of both tRNA and L1, and thecontrolled, driven motion of the subunits, ispredicted by the locking/unlocking model of ri-bosome function. According to this model, pep-tide bond formation unlocks the ribosome andtranslocation by EF-G locks the ribosome. Thismodel suggests two distinct conformations ofthe ribosome separated by the chemical eventsof peptidyl transfer and GTP hydrolysis, con-sistent with single-molecule data (Figure 5).

The ribosome begins a round of elongationin the locked, or nonrotated, conformation.In this state, the tRNAs are held firmly inthe classical configuration and the L1 stalk iskept open. Cognate tRNA selection at the Asite and subsequent peptide bond formationsignals ratcheting of the 30S subunit. In thisrotated conformation, both tRNA and the L1stalk are released and begin to fluctuate. Forcemeasurements indicate that peptide bondformation also weakens the ribosome:mRNAinteraction (73). Thus, controlled ratchetingweakens the interaction between the ribosomeand its ligands, preparing the ribosome totranslocate both mRNA and tRNA.

EF-G-driven translocation reverses the in-tersubunit conformation and moves the tRNAinto the P and E sites, where they areonce again held in the classical configuration.



Nonrotated Rotated

Figure 5A general model for ribosome dynamics and function. Interpretation of single-molecule dynamics datawithin the current locking/unlocking model of ribosome function. In the nonrotated state, tRNAs are heldin the classical configuration, and the L1 stalk is kept open. The stability of this conformation may preservereading frame and allow precise manipulation of tRNA during selection and catalysis. Ratcheting into therotated state upon peptide bond formation permits classical & hybrid and open & closed fluctuations of thetRNA and L1 stalk, respectively. These motions may facilitate movement of tRNA and mRNA duringtranslocation or initiation. The action of the GTPases IF2 and EF-G returns the ribosome to the nonrotatedstate. L1 opening during this transition may facilitate dissociation of the E-site tRNA.

To allow dissociation of the E-site tRNA, theL1 stalk is kept open. Having returned to thenonrotated state, the ribosome is once againlocked. In this model, the two global states ofthe ribosome—nonrotated (locked) and rotated(unlocked)—are distinguished by the strengthof ribosome:ligand interactions. The controlledtransition between these global states ensuresthat the specific nature of ribosome:ligand in-teractions is suited to the task at hand. Dur-ing tRNA selection and peptide bond forma-tion, the ribosome grasps its ligands firmly topreserve reading frame, monitor fidelity, andfine-tune catalysis. Prior to translocation, theribosome loosens its grip, allowing it the flex-ibility to shuttle the tRNA and step along themRNA.

Future PerspectivesFuture single-molecule translation studiespromise to further reveal the dynamic eventsunderlying the mechanism of translation bythe ribosome. Already, single-molecule tech-niques have succeeded in following transla-tion in real time. Single-molecule force mea-

surements have followed the ribosome as ittranslocates, revealing the molecular forcescontrolling this central event, whereas single-molecule FRET revealed the global dynamicsof the ribosome at each codon of the mRNA.These approaches will allow analysis of elon-gation events, such as ribosomal pausing, melt-ing of secondary structure, and frameshifting.New single-molecule fluorescence approachessuch as the use of zero-mode waveguides, whichtune the method to high concentrations (up to20 µM) of fluorescent ligands (56, 74), will al-low real-time monitoring of ligand interactionswith the ribosome during translation. These ex-periments would probe the relative timing offactor and tRNA interactions with the ribosomeduring initiation, elongation, and termination.

One goal of future studies will be to com-bine the conformational data obtained fromFRET measurements with the physical infor-mation provided by force techniques (35, 55).Compared with simpler systems, in which ei-ther force methods, as in the case of the ki-nesin molecular motor, or fluorescence meth-ods, as with RNA folding, have dominatedsingle-molecule studies, the complex nature of



translation has required application of bothmethods. Force/FRET methods would allowthe correlation of distinct ribosomal confor-mations with the position of the ribosome onthe mRNA. Also intriguing is the possibility ofdetermining simultaneously the stability andconformation of translational intermediates.Together, these data might explain how confor-mational transitions such as 30S ratcheting giverise to movement of the translational machin-ery. Other questions, such as the role played bylocal mRNA structure during translation, couldalso be assayed with a combined force/FRETapproach.

Future studies should apply single-moleculemethods to eukaryotic translation. Single-molecule studies of eukaryotic translationmight probe dynamic and uncharacterized pro-cesses such as scanning during initiation. Thisprocess is thought to deliver the eukaryoticPIC, assembled at the 5# end of the mRNA,to the start codon, setting the reading frameand signaling formation of the elongation-competent ribosome (38). The application ofsingle-molecule techniques to eukaryotic trans-lation will be challenging, but it is preciselythese types of systems that modern biophysicsmust tackle.

SUMMARY POINTS

1. Decades of biochemical and biophysical studies established the existing model of themechanism of translation.

2. This mechanism, which requires the ribosome to manipulate tRNA and step repetitivelyalong the mRNA, implies movement.

3. High-resolution structures of the ribosome and its ligands describe translation in atomicdetail, capturing the endpoints of large-scale rearrangements of the ribosome.

4. Direct observation of the dynamic events that underlie the mechanism of translation ischallenged by the complexity of translation.

5. Single-molecule methods, which eliminate ensemble averaging effects, have emerged aspowerful tools to interrogate translation.

6. Single-molecule fluorescence techniques have described the global dynamics of the ri-bosome, as well as the dynamic motions of the L1 stalk and tRNA.

7. Single-molecule force methods have directly probed the forces holding the ribosome onthe mRNA.

8. Recent applications of single-molecule force and fluorescence methods have allowed real-time observation of ribosome translocation and ribosome dynamics during translation.

FUTURE ISSUES

1. Real-time observation of single ribosomes will fully exploit the advantages of single-molecule techniques.

2. New technologies, such as zero-mode waveguides, will allow single-molecule techniquesto observe translation under more physiologically relevant conditions and permit theinterrogation of ribosome:ligand interactions.



3. The combination of fluorescence and force techniques, such as in force/FRET, willallow the observation of dynamic motions and the measurement of molecular forces tobe performed simultaneously.

4. The application of single-molecule techniques to eukaryotic translation systems willprovide new insight into currently uncharacterized dynamic events, such as scanning.

DISCLOSURE STATEMENTThe authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

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