translation at the single-molecule level...anrv345-bi77-09 ari 28 april 2008 11:49 contents...

ANRV345-BI77-09 ARI 28 April 2008 11:49

Translation at theSingle-Molecule LevelR. Andrew Marshall,1 Colin Echeverrıa Aitken,2

Magdalena Dorywalska,3 and Joseph D. Puglisi3,4

1Department of Chemistry, 2Biophysics Program, 3Department of Structural Biologyand 4Stanford Magnetic Resonance Laboratory, School of Medicine, StanfordUniversity, Stanford, California 94305; email: [email protected],[email protected], [email protected], [email protected]

Annu. Rev. Biochem. 2008. 77:177–203

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev.biochem.77.070606.101431

Copyright c© 2008 by Annual Reviews.All rights reserved

0066-4154/08/0707-0177$20.00

Key Words

FRET, hybrid state, optical tweezers, protein synthesis, ribosome,tRNA selection

AbstractDecades of studies have established translation as a multistep, multi-component process that requires intricate communication to achievehigh levels of speed, accuracy, and regulation. A crucial next stepin understanding translation is to reveal the functional significanceof the large-scale motions implied by static ribosome structures.This requires determining the trajectories, timescales, forces, andbiochemical signals that underlie these dynamic conformationalchanges. Single-molecule methods have emerged as important toolsfor the characterization of motion in complex systems, includingtranslation. In this review, we chronicle the key discoveries in thisnascent field, which have demonstrated the power and promise ofsingle-molecule techniques in the study of translation.

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FurtherANNUALREVIEWS

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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 178The Translation Cycle . . . . . . . . . . . . 179The Structural Biology of the

Translation Machinery . . . . . . . . . 181Translation at the Single-

Molecule Level . . . . . . . . . . . . . . . . 181DESIGN OF SURFACE-BASED

TRANSLATION SYSTEMS . . . . . 183Labeling the Components

of Translation . . . . . . . . . . . . . . . . . 183Immobilization Strategies . . . . . . . . . 184Other Considerations . . . . . . . . . . . . . 186

tRNA DYNAMICS ON THERIBOSOME DURINGELONGATION . . . . . . . . . . . . . . . . . 186Dynamics Prior to Peptide

Bond Formation. . . . . . . . . . . . . . . 187Dynamics Post-Peptide Bond

Formation . . . . . . . . . . . . . . . . . . . . 191ORIGINS OF RIBOSOME

MOVEMENT ON mRNA . . . . . . . 193CONCLUSIONS AND

PERSPECTIVES . . . . . . . . . . . . . . . . 194

INTRODUCTION

Translation converts genetic information intoproteins that execute the myriad tasks neces-sary for life. It is estimated that, in the simplestprokaryotic organisms, nearly half the dryweight of the cell and more than 80% of its en-ergy are used to drive the synthesis of proteins(1). The central component in translation isthe ribosome, a massive (megadaltons), mul-tisubunit biomolecular machine. Both smalland large ribosomal subunits, which comprisestructured RNA and protein elements, arehighly conserved in all kingdoms of life (2–6). The ribosome is a processive enzyme re-sponsible for two major tasks during proteinsynthesis: the reading of the genetic code andthe catalysis of peptide bonds. Processivity re-quires a delicate balance between accuracy andrate during protein synthesis. Furthermore,

rapid and precise cellular response to outsidestimuli demands that translation be tightlyregulated. Misregulation of translation leadsto disease (7, 8). Many clinically importantantibiotics, including streptomycin, tetracy-cline, and gentamicin, target the ribosome andimpair its function (9, 10).

In addition to the ribosome, mRNA andtRNA make up the core components of thetranslational machinery. mRNAs, which varyin length up to thousands of nucleotides, reg-ulate protein synthesis via both sequence andstructurally encoded elements (11–13). To de-cipher the genetic code, the ribosome em-ploys aminoacyl-tRNAs. The ribosome pro-vides tRNA access to mRNA codons viathree unique tRNA-binding sites: amino-acyl (A site), peptidyl (P site), and exit (Esite). The P site, which contains the catalyticsite for peptide bond formation, preferentiallybinds peptidyl-tRNA, whereas the A and Esites bind incoming aminoacyl- and exitingdeacylated tRNAs, respectively. The interplaybetween the ribosome, mRNA, and tRNAduring protein synthesis is central to themechanism of translation.

In vivo, ribosomes are estimated to syn-thesize between 6 and 20 peptide bonds persecond, while misincorporating less than 1 in1000 amino acids (14, 15). To achieve this bal-ance of speed and fidelity, the ribosome worksin concert with a variety of protein factors.Translation factors modulate the energeticsof translation, interacting directly with rRNA,ribosomal proteins, or both. Several key trans-lation factors use chemical energy from ATPor GTP hydrolysis to drive conformationalrearrangements, which are thought to lever-age the remodeling of ribosome structure togenerate macromolecular motion.

A subset of translation factors is highlyconserved in prokaryotes and eukaryotes,suggesting similarity in the basal mecha-nism of translation. In eukaryotes, however,where translation regulation is more preva-lent, the number of factors is greater (16).Numerous eukaryotic factors are the down-stream targets of kinase-driven regulatory

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pathways; modification of these factors tunesgene expression levels (17, 18). Communi-cation between translation factors, the ribo-some, tRNA, mRNA, and other cellular path-ways is the mechanistic basis for the high levelsof control that exist in translation.

A rich history of mechanistic studies hasunraveled the essential process of protein syn-thesis. Structural biology has described theribosome and its associated translation fac-tors in atomic detail. The remaining frontierin studies of the ribosome and translation isdynamics. Here, we review the use of single-molecule approaches to link mechanistic andstructural studies to provide novel views of thetranslation mechanism.

The Translation Cycle

Decades of genetic, biochemical, and bio-physical characterization have established ourcurrent understanding of translation as a com-plex, multistep, multicomponent process thatrequires a high level of regulation. Translationcan be parsed into three distinct stages: initia-tion, elongation, and termination. Each stagerequires the coordination of multiple compo-nents of the translational machinery and theprecise timing of molecular events (Figure 1).

Initiation is largely believed to be the rate-limiting and most regulated step in translation(19–22). In prokaryotes, where transcriptionand translation are coupled, interaction be-tween rRNA of the small ribosomal subunitand the Shine-Dalgarno (SD) sequence at the5′ end of an mRNA determines the appro-priate start site and reading frame for proteinsynthesis (23). The three initiation factors—IF1, IF2, and IF3—modulate the affinity ofthe P site for initiator tRNAfMet and tune therate of initiation complex assembly to ensureaccurate priming of protein synthesis (24, 25).In vivo, it has been estimated that initiationoccurs at a rate of 2.8 μM−1 s−1, which trans-lates into a delay time of approximately 2 sbetween initiation events on a single mRNA(26). Notably, initiation in vivo is several or-ders of magnitude faster than in vitro.

SD: Shine-Dalgarno

Cognate:Watson-Crick basepairing betweencodon and anticodonat nonwobblepositions, in additionto appropriatepairing at wobblepositions

Initiation on canonical eukaryoticmRNAs, which do not have an SD sequence,occurs via a distinct pathway and requiresmore than 11 protein factors (20, 21, 27–31).The process is distinguished by an energy-dependent mRNA-scanning step whereby apre-initiation complex assembled at the 5′

end travels upstream to locate an appropriatetranslation start site (32, 33). Evidence alsosuggests that the 5′ and 3′ ends of eukaryoticmessages communicate, perhaps linkingtermination with reinitiation on a singlemRNA (34). Eukaryotic translation initiationis tightly regulated by a number of cellularpathways responsible for cellular growth,proliferation, viral infection, and apoptosis(13, 35–37).

Following initiation, the assembled ribo-somal complex enters the elongation cycle,a repetitive process during which aminoacyl-tRNAs are decoded and peptide bonds aresynthesized (38–43). The ribosome selectstRNA via a two-step mechanism facilitatedby a ribosome-activated GTPase, elongationfactor Tu (EF-Tu, or eEF1A, in eukaryotes).During initial selection, a ternary complexof aminoacyl-tRNA, EF-Tu, and GTP bindsthe ribosome reversibly. Upon cognate tRNArecognition, GTP hydrolysis and dissocia-tion of EF-Tu permit the accommodation ofaminoacyl-tRNA into the A site, which leadsto peptide bond formation. Consistent withthe estimated rate for peptide bond synthesisin vivo, extensive biochemical data have es-tablished that individual events during tRNAselection occur on the millisecond timescale(44–46). Following peptide bond formation,elongation factor G (EF-G, or eEF2, ineukaryotes) catalyzes the rapid and coordi-nated movement of A- and P-site tRNAs, andtheir respective codons, in a process termedtranslocation (39, 41, 47). Several antibioticclasses, such as aminoglycosides, macrolides,and tetracyclines, target the prokaryotic ri-bosome and disrupt the rate and fidelity ofelongation.

When the ribosome encounters a stopcodon, the nascent peptide is released, and

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Initiation

Termination

Recycling

A

GTP

GTP

GTP

TuG

GDP

GTP

G

GTP

GDPGTP

GDP

1/2

Elongation

GTP

TuGTP

Tu

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GTP

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GDP

3

GTP

GDP

GDP

3

GDP

RRF

G

GTP

GTP

GDP2

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RRF

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3

GDP

GTP

GTPGDP

PE

G

3

Key:

Initiation factors

Stop codon

30S subunit

50 S subunit

Elongation factors

Ribosomal recycling factor

1 2

G

RRF

Tu

Release factors

1/2 3

Figure 1The prokaryotic translation cycle. Shown is the current model, which has been derived from biochemicaland biophysical studies. Initiation, mediated by initiation factors 1,2, and 3 ( green-shaded circles),culminates in the joining of 30S ( gray) and 50S (purple) subunits on the mRNA message primed withinitiator tRNA ( gray line with red circle) in the P site. This complex, aided by the elongation factors Tuand G (blue-shaded circles), subsequently undergoes multiple rounds of elongation. Termination, under thecontrol of release factors 1, 2 and 3 (red-shaded circles), frees the newly synthesized polypeptide uponrecognition of the stop codon. Ribosomal recycling factor ( yellow circle) and elongation factor G thenprepare the translational machinery for subsequent initiation events. Abbreviations: A, ribosomal A site;E, ribosomal E site; G, elongation factor G; P, ribosomal P site; RRF, ribosome recycling factor; Tu,elongation factor Tu.

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the ribosome is disassembled, setting the stagefor further rounds of protein synthesis (48–51). In prokaryotes, stop-codon recognitionand peptide release, known together as ter-mination, are facilitated by codon-specific re-lease factors RF1 or RF2. Evidence suggeststhat the peptidyl transferase center is respon-sible for the chemistry of peptide release(52). The activity and dissociation rates ofRF1 and RF2 are stimulated by the GTPaseRF3, which undergoes GDP-to-GTP ex-change upon ribosome binding (53, 54). Fol-lowing peptide release, ribosome recyclingfactor, in concert with EF-G and IF3, stim-ulates the dissociation of ribosomal subunits,deacylated tRNA, and mRNA to recycle thetranslational machinery (55). In contrast, eu-karyotes possess a single release factor, eRF1,which catalyzes peptide release from all stopcodons (56, 57). The function of eukaryoticrelease factor 3 (eRF3), a GTPase analogousto RF3, is unknown (27). In general, themechanism of recycling in eukaryotes remainsuncharacterized; however, a litany of play-ers has been implicated (27). As mentionedabove, recycling may also be directly linked toinitiation through an mRNA circularizationmechanism.

The Structural Biologyof the Translation Machinery

Breakthroughs in cryo–electron microscopyand X-ray crystallography have detailed thecomplexity of ribosome structure and its inter-actions with several components of the trans-lational machinery at atomic resolution (3, 5,6, 58–65). Interpretation of genetic, biochem-ical, and biophysical data within this structuralframework has underscored the role of rRNAin ribosomal function and has provided themeans to understand translation at the molec-ular level.

High-resolution structures reveal thesheer size of the translational machinery.The 250-A-diameter ribosomal particle or-chestrates the interaction of macromoleculartRNAs and protein factors on an mRNA tem-

plate. The active sites responsible for decod-ing mRNA, activating ribosomal GTPases,and catalyzing peptide bond formation areseparated by at least 50 A. Likewise, eachtRNA-binding site is separated from its neigh-bor by roughly 40 A (66). Yet the ribosomemust facilitate communication between thesedistinct regions. Structures of the ribosomein complex with various ligands reveal an in-tricate web of RNA-RNA, RNA-protein, andprotein-protein interactions, providing directlinks to the mechanisms of tRNA selection,GTPase activation, and peptide bond forma-tion.

These ribosome structures suggest thatlarge conformational rearrangements under-lie the mechanism of various steps in the trans-lation cycle. Crystal structures of EF-Tu andeukaryotic initiation factor 5B (eIF5B, a IF2homolog) in GTP- and GDP-bound formsdemonstrate the large domain-specific mo-tion that occurs after GTP hydrolysis (67–69).During elongation, tRNAs must be shuttledthrough the A, P, and E sites in a directionalmanner, requiring highly synchronized, large-scale movements of both tRNAs and mRNAs(66). Direct comparison of free and tRNA-bound ribosome structures reveals significantremodeling of both the local and global ar-chitecture of the small subunit upon tRNAbinding (70–73). Moreover, comparison ofcryo–electron microscopy structures of freeand EF-G-bound pretranslocation complexesidentifies a ratchet motion, resulting from thereorientation of the small subunit with respectto the large subunit (74). The function of thesemotions in processivity, fidelity, and regula-tion remains unknown.

Translation at the Single-Molecule Level

Translation is a complex process requiringintricate communication between multiplecomponents to achieve speed, accuracy, andregulation. High-resolution structural datahave provided a physical context for the ge-netic, biochemical, and biophysical models of


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FRET: fluorescenceresonance energytransfer

translation mechanism. Structures of the ribo-some in complex with its ligands suggest thatlarge-scale motions are at the root of impor-tant mechanistic events in translation. How-ever, the nature, timescale, and magnitude ofthese movements remain undetermined andtheir functional significance unknown.

Single-molecule spectroscopic and force-based techniques have emerged as power-ful tools for observing dynamic structuralrearrangements in multistep, multicompo-nent biological processes. These ultrasensi-tive techniques allow for observation of dis-tance changes on the subnanometer scaleand measurement of molecular forces inthe piconewton (pN) range (75–77). Single-molecule methods, which correlate chemi-cal and physical events with biological mo-tion, have probed mechanoenzymes (78, 79–84); nucleic acid processing enzymes, includ-ing RNA and DNA polymerases (85–92), nu-cleases (93), helicases (94, 95), gyrases (96,97), and topoisomerases (98); and the multi-step folding and unfolding trajectories of pro-teins and RNA (99–101). The principal ad-vantage of single-molecule observation is theelimination of the temporal- and population-averaging characteristics of bulk ensembles.Single-molecule data can be sorted and syn-chronized, thus removing these averaging ef-fects. As a result, single-molecule techniquescan directly characterize transient and rareevents, as well as parallel reaction pathways,all of which are not easily assayed in hetero-geneous and asynchronous ensembles. Theapplication of single-molecule techniques tothe study of protein synthesis has great po-tential to assist in building our understandingof translation and its regulation.

Single-molecule fluorescence methodsharness the sensitivity of fluorophores to theirenvironment to report on distance and ori-entation changes in biological systems (76,77, 102). One of the most powerful em-bodiments of single-molecule fluorescenceis total-internal-reflection-based fluorescenceresonance energy transfer (FRET). Total-internal-reflection illumination of donor flu-

orophore, which greatly improved signal-to-background ratios, results in dipolar energytransfer to an acceptor (103). The efficiencyof FRET is most sensitive to the distancebetween fluorophore dipoles (R), with a de-pendence of R−6. By following changes inFRET efficiency of single molecules in realtime, a unique perspective on the biomolec-ular events that make up complex biologicalpathways is provided.

FRET experiments are particularly adeptat characterizing dynamic conformationalchange when dye labeling is guided by high-resolution structures. Direct conversion ofFRET efficiency to absolute distances is com-plicated by the dependence of energy transferupon relative orientation of the fluorophores.Consequently, distance changes monitored byFRET are used qualitatively and validatedthrough other structural methods when fea-sible. Commonly used fluorophores and cur-rent detection technology allow for monitor-ing of distance changes on the order of 2–8nm, with millisecond time resolution, whichis optimal for observation of conformationalchanges on the timescales relevant to transla-tion (76, 77, 102, 104).

Direct manipulation of single moleculesby force probes the thermodynamic and ki-netic origins of motion in complex biolog-ical systems. Single-molecule manipulationcan be achieved by several methods, includingatomic force microscopy, magnetic tweezers,and optical tweezers (75, 105–107). Opticaltweezers, a methodology recently applied inthe study of protein synthesis, rely on the ra-diative pressure exerted by a tightly focusedlaser beam to capture small dielectric beadsattached to the biomolecule of interest. Themolecule is also attached to a surface or abead held by a second optical tweezer or by amicropipette tip. This allows for the applica-tion of force in a constant, gradient, or jumpmanner. In principle, optical tweezer meth-ods directly assay the forces required to inter-rupt macromolecular interactions, analogousto thermal melting in bulk. Additionally, ex-ternal forces impact the kinetics of molecular

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motion by perturbing the energy barriers andlandscapes. Optical tweezers have been usedto characterize complex folding trajectoriesfor RNA and proteins, the stochastic natureof transcription by RNA polymerase, and thewalking of myosin along actin filaments (78,79, 91, 108–111).

DESIGN OF SURFACE-BASEDTRANSLATION SYSTEMS

The challenges involved in observing trans-lation at the single-molecule level are mani-fold. Perhaps the most fundamental obstacleis to measure accurately and precisely the lightemitted from single fluorophores, displace-ments on the order of several angstroms, orpN forces exerted by single molecules. Suchmeasurements require gentle and specific at-tachment of reporter tags to the componentsof translation. Furthermore, immobilizationof the translational machinery broadens thescope of single-molecule experiments that canbe performed. Methodological advances inthe labeling, immobilization, and detection ofsingle molecules have been reviewed exten-sively elsewhere (76, 102–104, 112). In thissection, we focus on developments specific tothe study of translation.

Labeling the Componentsof Translation

Force methods require handles for thephysical manipulation and observation ofbiomolecules. Likewise, FRET techniquesrely on organic fluorophores, such as cya-nine or rhodamine derivatives, to report onlocation and conformational rearrangements.Several factors must be addressed when label-ing biomolecules for single-molecule study.

First, single-molecule experiments aremost insightful when labels are attached ina site-specific manner. High-resolution struc-tures of the ribosome and its components bestguide the choice of labeling site. Biomoleculelabeling is generally an iterative process,requiring optimization to enhance the ob-

served signal and to mitigate interference withbiomolecular structure or function. In opti-cal tweezer experiments, attachment must alsowithstand the applied forces. Typically, cova-lent attachment affords resistance to forces upto 1 nN, whereas specific noncovalent attach-ment schemes such as biotin-avidin withstandforces in the 10-to-300-pN range (75). Effortsin the specific labeling of tRNAs and the ri-bosome are discussed in more detail below.

tRNA labeling. General nucleic acid label-ing methodologies, such as 3′ or 5′ end la-beling, are not appropriate for tRNA becauseof the functional significance of its acceptorstem in aminoacylation and ribosome recog-nition. Therefore, internal labeling of tRNAsor amino acid labeling schemes are most fre-quently employed. One such strategy exploitsnaturally occurring modified nucleotides inEscherichia coli tRNAs (113–116). For single-molecule FRET studies, the 4-position ofthiouridine residues in the elbow region oftRNAfMet and tRNAPhe was covalently cou-pled to Cy3 and Cy5, respectively (117). X-raycrystallography demonstrates that the elbowpositions of A- and P-site tRNAs are sepa-rated by ∼40 A, which is optimal for probinginter-tRNA dynamics via FRET between Cy3and Cy5 (49). Importantly, modified tRNAsare fully active in aminoacylation, formyla-tion, complex formation with EF-Tu, and thestages of elongation (117). Alternatively, cou-pling of the α-amino group of fully chargedtRNA with amine-reactive dye conjugates al-lows for the direct probing of the puromycinreactivity of surface-bound ribosomes. Ow-ing to the high stringency of aminoacyl-tRNAsynthetases, the bulky dye species must be at-tached after aminoacylation (118).

Ribosome labeling. Although methodolo-gies for labeling of individual proteins or nu-cleic acids are well developed, their applica-tion in specific labeling of the ribosome iscomplicated by the high degree of conser-vation and structural complexity of the ribo-some. To date, two approaches have allowed


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the specific labeling of ribosomes. The firstuses reconstitution of ribosomes from la-beled components. The second allows di-rect labeling of mutant ribosomal particles bydye-labeled oligonucleotides. Each approachpresents distinct advantages and challenges.

In the first approach, ribosomal parti-cles are assembled in vitro from individuallypurified and selectively labeled components.This approach was initially used in bulk spec-troscopic studies where thiosemicarbazidederivatives of fluorescent dyes were directlyattached to the oxidized 3′ ends of the threeisolated rRNAs, followed by reassembly ofthe ribosomal subunits by incubation withextracted ribosomal proteins (119). A sim-ilar method was described more recentlyfor tagging ribosomal proteins modifiedby site-directed mutagenesis to containa single surface-exposed cysteine residuethat could be reacted with a fluorophoremaleimide conjugate (120, 121). Labeled re-combinant proteins were then incorporatedinto the 30S subunit by sequential additionof all the subunit components (121), and intothe 50S subunit by incubation with a sub-stoichiometric amount of protein-deficient50S particles isolated from a knockout strain(120). The major disadvantage of in vitroreconstitution-based approaches is the in-herent heterogeneity of the sample and theassociated loss of translational activity. Ri-bosomal subunits reassembled with labeledrRNAs or proteins are typically 35%–80%as active in polyuridine-directed polyphenyl-alanine synthesis as are wild-type ribosomesand show approximately 60%–70% tRNA-binding efficiency and 75% translocationefficiency as compared with wild-type trans-lation complexes (119–121). The reconsti-tution protocol relies on the viability ofthe required mutant and knockout ribosomalprotein strains.

An alternative strategy was recently de-veloped for high-efficiency tagging of rRNAvia oligonucleotide hybridization (122). Inthis approach, fluorescently labeled syntheticoligonucleotides are annealed to helical exten-

sions engineered into surface-accessible andphylogenetically variable regions of rRNA.The mutant ribosomes are assembled in vivoand labeled after isolation of intact particlesfrom viable strains expressing exclusively themodified rRNA. Optimization of the hair-pin extension sequence provided a highly sta-ble (Kd ∼ nM) and specific interaction withthe target oligonucleotide. The hybridizationprotocol yielded homogeneous populations oflabeled ribosomes that retained more than90% activity in translating a full-length pro-tein as compared with wild-type ribosomesand showed tRNA-binding and translocationefficiencies comparable to those of wild-typeribosomes. The primary limitation of thismethod comes from the high degree of phy-logenetic conservation within functional re-gions of rRNA, where any modification re-sults in a lethal phenotype. For this reason,the 50S subunit has proven more difficult tomodify and label than the 30S subunit (M.Dorywalska & J.D. Puglisi, unpublished ob-servations).

Immobilization Strategies

Considerable progress has been made inthe immobilization of biologically activesystems for biophysical analyses. Pavlov andcolleagues accomplished the first immobi-lization of an actively translating system,which they used to study ribosome assemblyon surface-coupled mRNAs (123, 124). Todate, several biologically active surface-basedtranslation systems have been designed.These systems permit the direct observationof time-evolving, multistep pathways duringtranslation, under both equilibrium andnonequilibrium conditions, on the secondsto minutes timescale.

Direct surface adsorption is the moststraightforward method for immobilizing thetranslational machinery. The negative chargedensity at the surface of untreated quartzlikely facilitates this nonspecific attachment,which is stable for long periods of time and canwithstand significant forces (75). Nonspecific

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adsorption suffers from the unknown modeof attachment and resulting random orienta-tion of immobilized molecules. Each of theseunknowns can complicate the interpretationof single-molecule measurements and renderbiomolecules inactive.

Despite these drawbacks, physical adsorp-tion allowed the first observations of sin-gle, biologically active ribosomes immobi-lized on a surface. By immobilizing 70Scomplexes with an initiator tRNAfMet la-beled at the α-amino group with tetramethyl-rhodamine, Sytnik et al. characterized theproperties of individual dye-labeled tRNAs(125). To probe the identity of the im-mobilized complexes, their reactivity withpuromycin was tested. Puromycin, a small-molecule mimic of aminoacyl-tRNA, reactsspecifically with acylated-tRNA in the Psite of the ribosome. Sytnik et al. demon-strated that the number of fluorescent tRNAsdecreased upon the addition of puromycinto the immobilized complexes, consistentwith tetramethylrhodamine-Met-puromycinrelease from the ribosome upon reaction withpuromycin.

Ribosomes adsorbed onto surfaces arecapable of performing multiple roundsof elongation (126). Biochemical charac-terization of immobilized 70S ribosomecomplexes demonstrated the activity ofsurface-bound ribosomes in the translationof a polyuridine message, albeit at a muchslower rate than observed in bulk. To observedirectly translation by single ribosomes, 3′-biotinylated polyruidine RNAs were boundto fluorescent neutravidin-coated beads. Thebead-polyuridine conjugates were then boundto surface-immobilized 70S ribosomes, whichrestricted the diffusion of the beads. Upon ad-dition of the factors necessary for polyphenyl-alanine synthesis, the beads showed a time-dependent reduction in their diffusion,consistent with a decrease in the length ofpolyuridine message between the bead andimmobilized ribosome. Interestingly, the rateof peptide bond formation measured fromsingle ribosomes is at least twofold faster

than that determined from bulk biochemicalcharacterization of surface-immobilized ribo-somes. This suggests heterogeneous activityin the ensemble of surface-immobilized ri-bosomes, which may be an intrinsic propertyof purified ribosomes or the result of theimmobilization strategy applied here.

Recent work demonstrated the feasibilityof covalent immobilization schemes. Vanziet al. developed a strategy for covalentlylinking ribosomes to surfaces in a nonspe-cific fashion through surface-exposed cysteineresidues of ribosomal proteins (127). Thismethodology requires the functionalizationof a glass coverslip with an amine-derivatizedsilane. Reaction of the amine-coated surfacewith heterobifunctional cross-linking agentsattaches a sulfhydryl-reactive maleimide. Cys-teine residues on ribosomal proteins reactwith surface-attached maleimide groups. Ri-bosomes immobilized in this fashion retaintheir ability to bind and translate polyuridinemessages.

An ideal surface-immobilization strategyfor the study of translation via single-moleculemethods would allow for specific, long-lived,and reproducible surface attachment withminimal nonspecific binding of ribosomes,tRNA, mRNA, and translation factors. Thismethodology would be flexible, allowing forthe attachment of any of the aforementionedcomponents for observation in force, fluo-rescence, or simultaneous force/fluorescencesingle-molecule experiments. Furthermore,the activity of translation observed free insolution would be recapitulated when com-ponents were surface immobilized. Satisfac-tion of all these requirements is a significantchallenge.

Blanchard and colleagues developed a sys-tem, which meets a number of these crite-ria (117). By adapting a quartz-surface prepa-ration strategy originally applied to DNAhelicases (95), Blanchard et al. developed ahighly active surface-based translation sys-tem, which has been successfully applied influorescence and force measurements (117,128, 129). This system takes advantage of the


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PEG: polyethyleneglycol

high-affinity biotin-streptavidin interactionand the antifouling properties of polyethyleneglycol (PEG) to achieve specific immobiliza-tion of ribosome complexes with low back-ground binding. Like the method proposed byVanzi et al., the quartz surface is first deriva-tized with a commercially available amino-functionalized silane. Treatment of these sur-faces with amine-reactive PEGs (5000 MW),a fraction of which are biotinylated, createsa PEG-coated surface that can be furtherderivatized with streptavidin. Streptavidinbound to surface-attached, biotinylated PEGis then used to tether 5′-biotinylated mRNAin association with 70S-initiated complexes tothe surface. The observed biotin-dependentbinding specificity is on the order of 500to 1 (117). A modified surface preparation,which relies on nonspecifically immobilizedbiotinylated BSA, has been employed in opti-cal tweezer-based force measurements (129).

Ribosomes immobilized on a PEG sur-face maintain their bulk-solution activity. 70Sinitiation complexes react with puromycinat the same rate as those free in solu-tion (117). Additionally, puromycin reactiv-ity established the competence of surface-immobilized ribosomes in ternary complexbinding and EF-G-catalyzed translocation.Direct observation of ternary complex bind-ing was achieved through stopped-flow deliv-ery of Cy5-labeled Phe-tRNAPhe to surface-immobilized 70S complexes initiated with aCy3-elbow-labeled fMet-tRNAfMet. The ap-pearance of inter-tRNA FRET upon ternarycomplex binding confirms the bimolecularrate constant determined in bulk (45). Thismethodology has provided a flexible platformfor the study of tRNA dynamics and the na-ture of ribosome-mRNA interactions duringelongation, as discussed below.

Other Considerations

High-intensity laser illumination required forthe excitation of single fluorophores andmaintenance of optical traps adversely af-fects the stability of single fluorophores and

biomolecules. The organic fluorophores cur-rently employed in single-molecule studies ofbiological systems photobleach rapidly anddisplay stochastic intensity fluctuations onthe millisecond timescale. Additionally, pho-toinduced reactive species cause oxidativedamage to both dyes and biomolecules, im-pairing their function (130, 131). These phe-nomena complicate interpretation and limitobservation time in single-molecule exper-iments (132, 133). Molecular oxygen (O2)plays a prominent role in both. Enzymatic O2-scavenging systems are typically employedto improve dye and biomolecule stabilityin single-molecule fluorescence and opticaltweezer experiments. Recent improvementsin O2-scavenging systems provide stable fluo-rescence for ∼30–40 s at laser intensities thatpermit high signal-to-noise ratios (134, 134a).

Single-molecule detection schemes re-quire flexible strategies for the labeling andimmobilization of biomolecules. Site-specificattachment of fluorescent labels on the com-ponents of translation allows for the powerof single-molecule FRET to be fully real-ized. Similarly, the specific surface immobi-lization of ribosomes via covalent or noncova-lent means is a versatile strategy in the designof biologically active surface-based translationsystems. Continued improvements in molecu-lar tagging and surface chemistry methodolo-gies will provide the flexibility to study morecomplex events in translation with greaterdetail.

tRNA DYNAMICS ON THERIBOSOME DURINGELONGATION

During elongation, the ribosome repeatedlyselects an appropriate aminoacyl-tRNA froma pool of more than 40 tRNAs and catalyzespeptide bond formation. The events leadingup to peptide bond formation dictate the fi-delity of peptidyl transfer, whereas the eventsthat follow ensure reading frame mainte-nance. The origin and nature of tRNA move-ment during elongation remain unexplained.

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Characterizing the dynamic trajectories oftRNAs on the ribosome during tRNA se-lection and translocation is crucial to un-derstanding the mechanism of each of theseprocesses.

Dynamics Prior to PeptideBond Formation

Discrimination between cognate, near-cognate, and noncognate tRNA cannotbe explained by free-energy differences incodon-anticodon base pairing only. Instead,a kinetic scheme has been proposed inwhich irreversible phosphate bond hydrolysisseparates two distinct stages: initial selectionand proofreading (135, 136). Biophysicaland biochemical studies have validated andprovided a detailed model for this scheme,highlighting the role of induced fit in tRNAselection (40, 59, 137, 138).

During initial selection, a ternary com-plex binds the ribosome through contactswith the large and small subunits. ProteinsL7 and L12 mediate the interaction withthe large subunit, while interaction with thesmall subunit is localized to rRNA, protein,and mRNA in the A site (139, 140). Near-cognate tRNAs dissociate at a 350-fold fasterrate than cognate tRNA at this stage (46).Changes in ribosome structure resulting fromcorrect codon-anticodon pairing suggest aninduced-fit mechanism (59). Recognition ofcognate tRNA leads to a 50,000-fold accel-eration in EF-Tu-catalyzed GTP hydroly-sis (43). The mechanistic details surround-ing the long-range signaling responsible forGTPase activation are not fully understood.Remodeling of intersubunit interactions andconformational changes within the ternarycomplex have been implicated in GTPase ac-tivation (40). GTP hydrolysis causes a dra-matic change in EF-Tu structure, decreasingits affinity for aminoacyl-tRNA and leadingto its dissociation from the ribosome. Sub-sequently, codon-dependent proofreading oc-curs, priming cognate tRNA for full accom-modation into the A site and peptide bond

Near-cognate: asingle mispair at onenonwobble position

Noncognate:mispairing at bothnonwobble positions

Postsynchroniza-tion: chronologicalalignment ofmultiple fluorescencetrajectories to areference event

formation. As with initial selection, induced-fit mechanisms may underlie the concomitantacceleration of cognate tRNA accommoda-tion and near-cognate tRNA dissociation dur-ing proofreading (40, 43).

Blanchard et al. observed tRNA selectionand peptide bond formation in real time at thesingle-molecule level (128). A ternary com-plex with Cy5-elbow-labeled Phe-tRNAPhe

was delivered to surface-immobilized 70Scomplexes with P-site-bound Cy3-elbow-labeled fMet-tRNAfMet. Postsynchronizationand superposition of individual fluorescencetrajectories revealed that tRNAs transitionrapidly to a high-FRET state via low- andmid-FRET states (Figure 2). Structural in-sights and biochemical tools aided assignmentof the low-, mid-, and high-FRET states toinitial tRNA binding, GTPase activation, andfull accommodation, respectively. The tran-sit time to high FRET was on the orderof 100 ms, which agrees with biochemicalmeasurements for the rate of accommodation(43). Analysis of progression from low to highFRET in the context of the current modelfor tRNA selection demonstrated the role oftRNA dynamics in decoding.

The ternary complex initially binds the ri-bosome through contacts with L7 and L12.Owing to the current labeling scheme, thisstep is invisible because the distance be-tween P-site tRNA and the incoming tRNA is>100 A. Blanchard and coworkers observedshort-lived transitions to low FRET (τ ∼50 ms), which were interpreted as A-site sam-pling by incoming cognate and near-cognatetRNAs (128). Excursions to low FRETare completely eliminated for noncognatecodons. Therefore, ternary complex bind-ing has two components: codon-independentbinding to L7/L12 and codon-dependentsampling of the A site. Rapid and reversiblecodon sampling is a novel observation identi-fied only via single-molecule techniques.

Following ternary complex binding, ini-tial tRNA selection occurs, preparing EF-Tu for GTP hydrolysis. Cognate tRNAdelivered to surface-immobilized ribosomes


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Raw fluorescence trajectories

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Figure 2Single-molecule tRNA dynamics. (a) Donor and acceptor fluorescence intensity traces were collectedfrom hundreds of individual ribosome complexes. Calculated fluorescence resonance energy transfer(FRET) traces were subsequently superimposed to visualize ensemble behavior. Postsynchronizationto the initial appearance of FRET revealed the chronological trajectory of FRET changes.(b) Postsynchronized color overlays under various conditions identified three distinct FRET states, whichwere assigned with the aid of existing biochemical and structural data. Analysis of individual state lifetimes,as well as the ensemble progression through these states, provides new insight into the nature of tRNAselection. (c) Interpretation of FRET trajectories in the context of high-resolution structural data providesa dynamic model for tRNA selection. Abbreviation: GDPNP, guanosine 5′-[β,γ-imido] triphosphate.

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transition to mid- then high FRET. Thenonhydrolyzable GTP analog, guanosine 5′-[β,γ-imido] triphosphate (GDPNP), stallsthis progression at a long-lived mid-FRETstate (τ ∼ 8 s), indicating that transition fromlow to mid-FRET occurs prior to GTP hy-drolysis; near-cognate tRNAs are preferen-tially rejected prior to reaching mid-FRET.Therefore, this transition represents initialtRNA selection. Discrimination at this stepfavors cognate tRNA sixfold, which confirmsprior biochemical work (44, 46).

Individual FRET traces from GDPNP-stalled deliveries reveal both long- and short-lived transitions (τ ∼ 8 and τ < 0.5 s,respectively) to mid-FRET, which can beinterpreted as successful and unsuccess-ful GTPase activation attempts, respectively(141). The observation of these thermal tRNAfluctuations is unique to single-molecule ap-proaches. Comprehensive analysis of thesefluctuations indicates that cognate tRNAs notonly preferentially transit to mid-FRET, butthey also sample this state at a threefoldfaster rate than near-cognate tRNAs. Thisaccelerated progression of cognate tRNAthrough codon recognition compared withnear-cognate tRNA had not been observed inbulk (44, 46).

The mechanism whereby cognate tRNAselection activates EF-Tu for GTP hydrolysisis unknown. Structural and biochemical stud-ies have identified conserved rRNA and pro-tein elements that interact with the ternarycomplex and regulate GTPase activity, in-cluding the universally conserved sarcin-ricinloop (SRL) (142, 143). Cleavage of the SRLby toxins—sarcin, ricin, and restrictocin—inhibits tRNA delivery and translocation(144). In single-molecule tRNA-selection as-says, restrictocin cleavage of the SRL slowsprogress to high FRET 13-fold as comparedwith unmodified ribosomes (128). Transitto mid-FRET is unaffected by SRL cleav-age (Figure 3). This agrees with biochemi-cal evidence that implicates the SRL-ternarycomplex interaction in GTP hydrolysis (145).Furthermore, this points to tRNA motion

GDPNP: guanosine5′-[β,γ-imido]triphosphate

SRL: sarcin-ricinloop

during the low-to-mid-FRET transition as anallosteric link between decoding and GTPaseactivation.

Following GTP hydrolysis, EF-Tu under-goes a conformational change that releasesaminoacyl-tRNA to enter the A site. The sub-sequent proofreading step determines the fateof the remaining tRNA. For cognate tRNA,Blanchard et al. observed rapid progressionfrom mid- to high FRET (128). The FRETefficiency (∼0.75) for the high-FRET state isconsistent with the distance between the el-bow regions of A- and P-site tRNAs deter-mined by X-ray crystallography (146). More-over, progression to high FRET requiresGTP hydrolysis (128). It follows that thistransition represents completion of proof-reading and full accommodation in the A siteprior to peptide bond formation. The overallerror frequency for single ribosomes was de-termined to be ∼7 × 10−3, allowing for anestimation of the selection efficiency duringproofreading of ∼24 to 1.

Prior biochemical, biophysical, and struc-tural data have pointed to induced-fit mecha-nisms as the basis for discrimination in initialselection and proofreading. Cognate codon-anticodon interaction results in local andglobal changes in small-subunit architecture.rRNA residues, A1492 and A1493, in the de-coding site adopt an extrahelical conforma-tion to recognize codon-anticodon pairing,and the head and shoulder domains rotate to-ward the center of the small subunit uponcognate tRNA recognition (70, 140). Thesemovements are not observed for binding ofnear-cognate tRNA (71). Additionally, con-formational flexibility of the GTPase domainappears to play a role in positioning the in-coming ternary complex for rapid GTP hy-drolysis (143, 147). These structural rear-rangements decrease kinetic barriers to tRNAmovement and likely aid in trapping thermalfluctuations required to overcome these bar-riers during tRNA selection events.

Consistent with this view, single-moleculefluorescence techniques have yielded accel-erated rates for cognate tRNA progression


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to mid-FRET (GTPase activation) and fornear-cognate tRNA rejection prior to mid-FRET (128, 141). Furthermore, tRNA deliv-ery stalled at mid-FRET by GDPNP or SRLcleavage highlights the importance of stabiliz-ing contacts between the large subunit and theternary complex in facilitating GTPase acti-vation. Interestingly, these stalled complexesare not static and display transient fluctuationsto high FRET on the millisecond timescale.A-site sampling in these stalled complexessuggests further conformational changes in-volved in the full accommodation of tRNAthat occur after GTP hydrolysis.

Several antibiotic classes specifically tar-get the ribosome, interfering with tRNA se-lection; the effects of these drugs on ribo-some function have been monitored usingsingle-molecule FRET (Figure 3). Tetracy-cline, a potent inhibitor of tRNA delivery,hinders advancement to the mid-FRET state,resulting in FRET trajectories with numer-ous short-lived transitions to low FRET (128,148). Tetracycline affects neither the arrivaltime of FRET during ternary complex deliv-ery nor the delivery of near-cognate tRNA(128). Together with structural and biochem-ical data, these experiments implicate tetracy-cline in blocking tRNA motion necessary forselection (149, 150).

The aminoglycoside antibiotic paro-momycin causes rearrangements in smallsubunit architecture similar to those seenupon cognate tRNA binding, increasing thelikelihood that near-cognate tRNAs transitto the high-FRET state (151–154; S.C.Blanchard, S. Chu & J.D. Puglisi, unpub-lished observations). This confirms theclassification of paromomycin as a potent

miscoding agent and also points to induced-fit mechanisms in the initial selection oftRNA (155).

Gonzalez et al. observed that thiostrepton,a cyclic peptide antibiotic that binds in thevicinity of the GTPase center, decreases thefrequency of successful GTPase activation at-tempts (156). Thiostrepton is known to in-hibit the GTPase activity of each ribosome-associated GTPase and likely blocks stabledocking of the ternary complex at the GTPasecenter during tRNA selection (156–160). Thisresult, together with the previously observedinhibition of GTP hydrolysis by SRL cleav-age, supports a connection between ternarycomplex association at both the SRL andGTPase center and activation of EF-Tu.

Kirromycin, an antibiotic that targets EF-Tu, uncoupling GTP hydrolysis and confor-mational change, stalls tRNA delivery at themid-FRET state (128, 161–163). Kirromycinlikely inhibits the transit of tRNA to the Asite after GTP hydrolysis. Ribosome com-plexes stalled at mid-FRET with kirromycinundergo transient thermal fluctuations, whichresult in sampling of the high-FRET state(128). This points to additional induced-fit interactions during proofreading, drivenby the EF-Tu conformational change ordissociation.

Dynamics Post-PeptideBond Formation

Peptide bond formation transfers the nascentpeptide from P-site tRNA to A-site tRNA.For repetitive peptide bond formation tocontinue during elongation, A- and P-sitetRNAs must be shuttled to the P andE sites, respectively. To orient tRNA for

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Inhibitors of tRNA selection. The current model for tRNA selection, based on bulk biochemical andbiophysical studies, as well as on recent single-molecule results, is shown. Single-molecule studies haverevealed the mode of action of several inhibitors of tRNA selection. Both tetracycline and thiostreptonwere shown to inhibit initial tRNA selection. Tetracycline inhibits progression to the GTPase-activatedstate, likely via steric interactions, which hinder tRNA movement. Thiostrepton may bind at the GTPaseactivation center, in competition with the ternary complex. Restrictocin, an enzyme shown to cleave thesarcin-ricin loop, slows GTP hydrolysis. Kirromycin interferes with tRNA accommodation, likely byinhibiting conformational changes in EF-Tu.


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Hybrid state: astaggeredconfiguration oftRNAs on theribosome

Classical state: averticalconfiguration oftRNAs on theribosome

efficient peptidyl transfer and to maintain thereading frame, the ribosome makes specificinteractions with tRNA and mRNA (164–166). Translocation requires that these stabi-lizing interactions be altered to allow for theconcerted, large-scale movement of mRNAand tRNA. The mechanism whereby pep-tidyl transfer signals the cascade of subse-quent translocation events is a key area ofinvestigation.

tRNA selection culminates with peptidyl-tRNA and aminoacyl-tRNA adopting classi-cal positions in the P and A sites, respectively.This configuration is represented by the high-FRET state observed during tRNA deliveryexperiments. Biochemical studies have iden-tified a unique intermediate state for tRNAsfollowing peptide bond formation, known asthe hybrid state (167). In the hybrid state,interactions between the acceptor stems ofA- and P-site tRNAs and the large subunitare broken, and new contacts are establishedin the P and E sites, respectively, whereas theposition of codon-anticodon complexes re-mains unchanged. Hybrid-state formation islikely a native property of the ribosome, as itsformation is not dependent upon translationfactors. The acylation state of A-site tRNA,which affects hybrid-state formation, alsomodulates the rate and accuracy of transloca-tion, strongly implicating the hybrid state intranslocation (168, 169). Recently, the hybridstate was directly identified as an intermediatealong the pathway of EF-G-catalyzed translo-cation (170). However, owing to its transientnature, the hybrid state has eluded attemptsat high-resolution structure determination.

Single-molecule FRET experiments haverevealed the dynamic nature of the hybridstate. Following successful accommodation tothe high-FRET state in ternary complex de-livery experiments, a reversible transition to asecond mid-FRET state was observed (117).Delivery of the ternary complex to ribosomeswith deacylated tRNAfMet in the P site favoredthe high-FRET state without affecting thetrajectory of delivery prior to full tRNA ac-commodation. Therefore, transitions to mid-

FRET after successful tRNA selection resultfrom tRNA movement to a unique, peptide-bond-favored state, which was assigned as thehybrid state. This is consistent with biochem-ical and biophysical studies, which demon-strate that hybrid-state formation is preferredfor A-site-bound peptidyl-tRNA.

The dynamic equilibrium of classical andhybrid states is modulated by the nature oftRNA in the A site and [Mg2+]. In a studyby Kim et al., hybrid-state dynamics wereobserved in two ribosome complexes, whichdiffered only in the identity of A-site tRNAPhe

(171). In each complex, Cy3-labeled P-sitetRNAfMet was paired with either Cy5 Phe-tRNAPhe or N-acetylated Cy5 Phe-tRNAPhe,a peptidyl-tRNA analog in the A site. At[Mg2+] exceeding 10 mM, the classicalstate was predominant in both complexes,whereas decreasing [Mg2+] shortened theclassical-state lifetime, favoring hybrid-stateoccupancy for complexes with N-acetylatedtRNAPhe. A-site peptidyl-tRNA destabilizesthe classical state, without affecting the freeenergy of the classical/hybrid transitionstate, an effect magnified at low [Mg2+].Similarly, Munro et al. have demonstratedthat hybrid-state stability is dependent uponthe identity of A-site peptidyl-tRNA (172).

Munro et al. also employed improvedtime resolution, coupled with Markovmodel–based kinetic analysis, to identify andcharacterize a novel metastable hybrid state(172). This state, in which only the acceptorend of deacylated P-site tRNA adopts anE-site position, is in equilibrium with boththe classical and conventional hybrid states.High classical-state occupancy upon deletionof ribosomal protein L1—involved in E-siteremodeling—implies that each hybrid staterequires movement of the acceptor end ofP-site tRNA to the E site. Mutation of theA loop, which base-pairs with the acceptorend of A-site tRNA, favors formation ofthe conventional hybrid state with a vacant,large-subunit A site. Disrupting base pairinteractions between the P loop and P-sitetRNA results in predominant formation of

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the new hybrid-state intermediate with avacant, large-subunit P site. P-loop mutationalso reduces puromycin reactivity, consistentwith the predicted position of the new hybridstate. Munro et al. observed that independentmovement of P-site tRNA accounts for∼45% of hybrid-state excursions, openingthe possibility that translocation can proceedvia parallel pathways.

Single-molecule fluorescence studies havealso linked the disruption of tRNA dynamicswith impaired translocation. Deletion of L1,which strongly inhibits translocation (173),slows transitions to and accelerates transitionsfrom hybrid states (172). Likewise, viomycin,a translocation inhibitor that modulates inter-subunit dynamics (174), decreases the rate offluctuation between classical and hybrid states(171). These observations implicate ribosomedynamics in hybrid-state formation. As intRNA selection, single-molecule and bulk ob-servations are consistent with an induced-fitmechanism (172).

ORIGINS OF RIBOSOMEMOVEMENT ON mRNA

Whereas single-molecule fluorescence tech-niques have explored the role of tRNAdynamics in translocation, recent single-molecule force measurements have begun toprobe the structural basis for ribosome move-ment along the mRNA. Vanzi et al. employedoptical tweezer methods to measure the sta-bility of ribosome complexes on polyuridinetemplate (127). This study demonstrated theutility of force measurements in monitoringribosome-mRNA interactions.

Most bacterial mRNAs contain a purine-rich SD sequence, which interacts with theribosome, setting the start site and readingframe for translation. Upon peptide bond for-mation, the ribosome must translocate, thusdisrupting its interaction with the SD se-quence. This and subsequent translocationevents require precise regulation of structuralrearrangements that allow partial dissociationof the ribosome from mRNA and associated

tRNAs, while keeping the entire translationcomplex intact.

To understand the mechanistic interplaybetween the components involved in the pro-cessive movement of the ribosome, Uemuraand coworkers applied single-molecule op-tical tweezer methods to measure directlythe forces exerted between the ribosome andmRNA at different stages of the transla-tion cycle (129). Force measurements wereperformed on ribosomal complexes teth-ered to a streptavidin surface through a bi-otinylated mRNA, derived from a naturalT4 gene 32 mRNA, with or without astrong SD sequence. For laser trapping,antidigoxygenin-coated beads were conju-gated to a digoxigenin-tagged oligonu-cleotide hybridized to the ribosome via anextension engineered in the 16S rRNA (175).This permitted the measurement of theforce required to disrupt interactions withinthe complex (Figure 4). Control experimentsverified that rupture likely results from thedissociation of the ribosome from the mRNA.The measured rupture force is thus equivalentto the force with which the ribosome grips themRNA (129).

The authors measured the strength ofribosome-mRNA interactions for complexesassembled on SD-containing and SD-lackingmRNAs, in the context of different tRNAs.Binding of deacylated tRNAfMet to the ribo-somal P site stabilized the ribosome-mRNAcomplexes by ∼5 pN, and subsequent bindingof Phe-tRNAPhe to the A site further strength-ened the interaction by ∼10 pN. Althoughthe increase in rupture force upon addition oftRNAs was comparable for the SD-containingand SD-deficient complexes, the actual mea-sured force was ∼10 pN higher in the pres-ence of the SD interactions, indicating thatthe SD sequence plays a role in stabilizing theribosome on the mRNA. Importantly, the oc-cupancy of tRNAs in the P and A sites of indi-vidual ribosomal complexes was verified priorto each force measurement using FRET, al-lowing for elimination of partially dissociatedcomplexes from further experiments.


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Beadfluctuations

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Figure 4Single-molecule measurements of ribosome-mRNA forces. Theribosome-bead complex, tethered to the surface via mRNA, showsrandom fluctuations around the point of attachment. The fluctuationsbecome suppressed once the bead is trapped with optical tweezers. Next,movement of the microscope stage results in the gradual displacement ofthe bead from the trap center position, which can be correlated to anincreasing force exerted on the ribosomal complex. Once the externalforce exceeds the intermolecular forces within the complex, the complexdissociates, or ruptures, and the bead returns to the trap center position.Measurements are repeated multiple times under each experimentalcondition to obtain a population distribution of rupture forces.

Addition of N-acetyl-Phe-tRNAPhe, apeptidyl-tRNA analog, to the A site ofthe ribosome bound to the SD-containingmRNA resulted in a decrease in the ruptureforce of almost 14 pN, as compared with thecomplex with Phe-tRNAPhe in the A site. Asimilar result was obtained when ribosome-catalyzed peptide bond formation, resultingin a dipeptidyl-tRNA bound in the A site,was performed prior to force measurement.This is consistent with previous biochemicaland structural studies demonstrating that theribosome can sense the chemical identity ofthe tRNAs bound in the P and A sites (168,176–179). For the post-peptidyl transfercomplexes, the rupture forces measured inthe presence or absence of the SD sequencewere essentially the same, suggesting thatthe SD interactions are weakened followingthe formation of the first peptide bond (129).The destabilization of the ribosome-mRNAcomplex is likely an important aspect ofthe translocation mechanism, allowing themRNA to slide through the ribosomalintersubunit space to position the next codonin the A site.

Previous biochemical studies have impli-cated the SD sequence in both the stabi-lization of the ribosome-mRNA interactionand increasing the accuracy of translocation(177, 180). Single-molecule force measure-ments have provided a mechanistic basis forthese observations.

CONCLUSIONS ANDPERSPECTIVES

Single-molecule techniques have probed thedynamic nature of translation. Leveragingdecades of biochemical data and high-resolution structural information, single-molecule approaches have investigatedfundamental translation events such as tRNAselection and peptide bond formation. Thesemeasurements, performed on the prokaryotictranslation system, have yielded new insightsinto the fundamental mechanism of pro-tein synthesis. Mechanistic models derived

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from these single-molecule approaches aresubsequently testable by bulk methods.

In monitoring the dynamic conforma-tional rearrangements between P-site tRNAand incoming aminoacyl-tRNA, novel ob-servations of codon-anticodon sampling andunsuccessful attempts at GTPase activationwere made during initial tRNA selection (128,141). Additionally, authors have suggestedthat tRNA movement during initial selectionis the means through which codon recogni-tion signals GTPase activation. These discov-eries open several avenues for future researchvia single-molecule methods. For example,the current inter-tRNA-FRET approach pro-vides little insight into the nature of codon-independent ternary complex binding. Simi-larly, resolving the degeneracy of mid-FRETstates is crucial to understanding GTPase ac-tivation. Investigation of each will require dis-tinct labeling and experimental approaches.Furthermore, the rearrangements of the ribo-some and EF-Tu during tRNA selection areas yet uncharacterized. Single-molecule ob-servation may help unravel the contributionsof ribosome and factor dynamics in tRNAselection.

Single-molecule methods have also ex-plored the relationship between peptidyltransfer and hybrid-state formation and standpoised to address other aspects of transloca-tion (171, 172). Correlation of binding eventsand conformational changes with GTP hy-drolysis and peptide bond formation shouldprovide mechanistic insight into the regu-lation of translocation. With current label-ing schemes for ribosomal subunits, single-molecule fluorescence techniques may soonlink tRNA and ribosome dynamics (120–122).

To date, single-molecule observation ofprokaryotic translation has been limited toinitial steps in protein synthesis. The major-ity of experiments have employed an mRNA-bound ribosome containing tRNAfMet in the Psite. tRNA decoding and accommodation, aswell as hybrid-state dynamics, have been stud-ied by the addition of tRNAPhe and the requi-site protein factors. Multiple tRNA delivery,

peptide bond formation, and translocation cy-cles have not been observed. Although ini-tial tRNA accommodation and peptide bondformation are of unquestionable biologicalsignificance, they may be unique events. Sub-sequent rounds of translation involve the in-teraction of A-site tRNA with a P-site tRNA,bound to the growing polypeptide chain, aswell as E-site tRNA. Untangling this networkof interactions will provide insight into theorigins of ribosome processivity.

Translation also occurs in the presenceof myriad protein factors, many at relativelyhigh biological concentrations (μM). Themolecular complexity and high backgroundsignals resulting from a true translationsystem remain a challenge, although recenttechniques may alleviate these problems (181,182). Single-molecule fluorescence and forcemeasurements performed in this contextshould contribute a more representative un-derstanding of the dynamic events underlyingthe mechanism of protein synthesis.

Recent work has broadened the scope ofsingle-molecule FRET experiments; three-color FRET and combined force and flu-orescence measurements have allowed formore complex experimental design (183–186). Equally promising is the ability ofsingle-molecule techniques to time mechanis-tic events to the binding or release of variousspecies, such as protein factors. This obser-vation paradigm, which could be described aschronological bookkeeping, may yield pow-erful insight into the role of various proteinfactors.

These approaches can explore higher-levelregulation events in eukaryotic translation.Single-molecule force measurements have al-ready begun to explore helicase-assisted scan-ning by the small subunit (187). Event tim-ing by single-molecule fluorescence mightsucceed in cataloguing the assembly anddisassembly of regulatory components in-volved in subunit recruitment, pre-initiationcomplex rearrangement, internal-ribosome-entry-site-controlled initiation, eukaryoticinitiation factor 2 (eIF2)-mediated translation


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inhibition, and other important initiationevents. Single-molecule FRET measure-ments may also provide insight into large-scale conformational movements unique toeukaryotic translation, such as cap-dependentcommunication of the 5′ and 3′ ends of themRNA template or small-subunit scanning.Dynamic fluorescence tracking and FRET ex-periments may also be extended to study thefolding of single polypeptides in the ribosomalexit tunnel or chaperone interactions at thetunnel exit site. The ability of single-moleculetechniques to detect rare or transient inter-mediates, and to correlate these intermediateswith mechanistic events, provides a powerfultool for understanding the complex regulatoryprocesses controlling eukaryotic translation.

Many of these experiments are currentlyfeasible. Others, however, will requiretechnological and methodological improve-ments. Nonspecific adhesion of molecularcomponents to immobilization surfacescurrently limits system complexity in manysingle-molecule applications (112). Im-proved nonfouling surfaces would allowfor the addition of more diverse proteinand nucleic acid components. Surface im-provements may also decrease backgroundfluorescence, yielding greater sensitivity influorescence applications. The applicationof microfluidic technology may improvesolution control, mixing, and reproducibilityin single-molecule techniques, while alsoreducing background signals. Novel func-tionalization techniques may allow for moreefficient, specific, and less-invasive labelingof biomolecules. Improvements in dyestability will permit extended illuminationin fluorescence experiments, minimizing

confusing intensity fluctuations and allowingfor prolonged observation; recent work hasdemonstrated novel approaches for improveddye behavior (134, 134a). Also promising isthe introduction of statistical and pattern-matching algorithms for data analysis: HiddenMarkov models have recently been used tointerpret intensity fluctuations in single-molecule fluorescence experiments (188).

Improvements in technology and method-ology promise to broaden the power andscope of single-molecule studies of transla-tion. Recent work by Yu et al. suggests thatsingle-molecule techniques may also probeprotein synthesis in vivo (189). By fusing afast-maturing yellow fluorescent protein vari-ant to Tsr, a membrane protein, the authorswere able to visualize the production of indi-vidual protein molecules. Translation was ob-served to proceed in bursts, likely a result ofrare and transient dissociation of the lac re-pressor controlling expression of the tsr-venusgene. This method represents a novel tool forobserving the stochastic nature of protein ex-pression and heralds the possibility of real-time observation of cellular responses to en-vironmental changes and other stimuli (190).

Single-molecule observation illuminateshighly regulated, multistep, multicomponentpathways, including translation, from a novelperspective. These studies rely heavily uponexisting and future biochemical, biophysical,and structural information. Only in this con-text has single-molecule observation provenits power. Buttressed by sufficient biochemi-cal and structural data, single-molecule meth-ods can transform stop-animation models ofmechanism into directly observed, detailed,and dynamic movies of biochemical function.

SUMMARY POINTS

1. Single-molecule methods lift the constraints of temporal and population averagingthat have limited detailed analysis of dynamic processes in translation.

2. Development of highly active surface-based translation systems for single-moleculeapproaches requires methods for specific tagging and immobilization of the compo-nents of translation.

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3. Novel observations of codon-anticodon sampling and unsuccessful attempts atGTPase activation for near-cognate tRNA highlight the power of single-moleculeapproaches in revealing rare and transient events in translation.

4. tRNA movement that occurs during initial selection likely serves as the allosteric linkbetween codon recognition on the small subunit and GTPase activation on the largesubunit.

5. Induced-fit mechanisms during tRNA selection and GTPase activation are the targetof several antibiotics.

6. A novel hybrid state in which only deacylated P-site tRNA adopts a hybrid configu-ration has been identified.

7. Peptide bond formation destabilizes the interaction between the ribosome and theSD sequence of mRNA, suggesting communication between the peptidyl transferasecenter and the small ribosomal subunit during elongation.

FUTURE ISSUES

1. The power of single-molecule techniques has been well established in the study ofelongation in prokaryotic translation systems. The application of these techniques inthe study of initiation and termination, as well as in eukaryotic translation, is on thehorizon.

2. Inter- and intrasubunit dynamics during translation have been implicated in a numberof processes. Specifically labeled ribosomal subunits promise to elucidate the natureand timescale of subunit motions, as well as their correlation with other events intranslation.

3. Current study of translation via single-molecule techniques has been limited to initialtRNA accommodation and peptide bond formation events. The study of multiplerounds of elongation can report on the physical origins of processivity in translation.

4. Methodological advances aimed at improving biomolecule labeling, surface chemistry,fluorescent dye stability, and data analysis are required to broaden the scope of single-molecule translation studies.

5. Technological advances that have allowed for single-molecule observation of proteinsynthesis in vivo promise to elucidate the role of translation regulation in cellularresponse.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.


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Annual Review ofBiochemistry

Volume 77, 2008Contents

Prefatory Chapters

Discovery of G Protein SignalingZvi Selinger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

Moments of DiscoveryPaul Berg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 14

Single-Molecule Theme

In singulo Biochemistry: When Less Is MoreCarlos Bustamante � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 45

Advances in Single-Molecule Fluorescence Methodsfor Molecular BiologyChirlmin Joo, Hamza Balci, Yuji Ishitsuka, Chittanon Buranachai,and Taekjip Ha � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 51

How RNA Unfolds and RefoldsPan T.X. Li, Jeffrey Vieregg, and Ignacio Tinoco, Jr. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 77

Single-Molecule Studies of Protein FoldingAlessandro Borgia, Philip M. Williams, and Jane Clarke � � � � � � � � � � � � � � � � � � � � � � � � � � � � �101

Structure and Mechanics of Membrane ProteinsAndreas Engel and Hermann E. Gaub � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �127

Single-Molecule Studies of RNA Polymerase: Motoring AlongKristina M. Herbert, William J. Greenleaf, and Steven M. Block � � � � � � � � � � � � � � � � � � � �149

Translation at the Single-Molecule LevelR. Andrew Marshall, Colin Echeverría Aitken, Magdalena Dorywalska,and Joseph D. Puglisi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �177

Recent Advances in Optical TweezersJeffrey R. Moffitt, Yann R. Chemla, Steven B. Smith, and Carlos Bustamante � � � � � �205

Recent Advances in Biochemistry

Mechanism of Eukaryotic Homologous RecombinationJoseph San Filippo, Patrick Sung, and Hannah Klein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �229

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Structural and Functional Relationships of the XPF/MUS81Family of ProteinsAlberto Ciccia, Neil McDonald, and Stephen C. West � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �259

Fat and Beyond: The Diverse Biology of PPARγ

Peter Tontonoz and Bruce M. Spiegelman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �289

Eukaryotic DNA Ligases: Structural and Functional InsightsTom Ellenberger and Alan E. Tomkinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

Structure and Energetics of the Hydrogen-Bonded Backbonein Protein FoldingD. Wayne Bolen and George D. Rose � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �339

Macromolecular Modeling with RosettaRhiju Das and David Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �363

Activity-Based Protein Profiling: From Enzyme Chemistryto Proteomic ChemistryBenjamin F. Cravatt, Aaron T. Wright, and John W. Kozarich � � � � � � � � � � � � � � � � � � � � � �383

Analyzing Protein Interaction Networks Using Structural InformationChristina Kiel, Pedro Beltrao, and Luis Serrano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �415

Integrating Diverse Data for Structure Determinationof Macromolecular AssembliesFrank Alber, Friedrich Förster, Dmitry Korkin, Maya Topf, and Andrej Sali � � � � � � � �443

From the Determination of Complex Reaction Mechanismsto Systems BiologyJohn Ross � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �479

Biochemistry and Physiology of Mammalian SecretedPhospholipases A2

Gerard Lambeau and Michael H. Gelb � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �495

Glycosyltransferases: Structures, Functions, and MechanismsL.L. Lairson, B. Henrissat, G.J. Davies, and S.G. Withers � � � � � � � � � � � � � � � � � � � � � � � � � � �521

Structural Biology of the Tumor Suppressor p53Andreas C. Joerger and Alan R. Fersht � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �557

Toward a Biomechanical Understanding of Whole Bacterial CellsDylan M. Morris and Grant J. Jensen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �583

How Does Synaptotagmin Trigger Neurotransmitter Release?Edwin R. Chapman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �615

Protein Translocation Across the Bacterial Cytoplasmic MembraneArnold J.M. Driessen and Nico Nouwen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �643

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Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms,Connected Processes, and DiseasesRoland Lill and Ulrich Mühlenhoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �669

CFTR Function and Prospects for TherapyJohn R. Riordan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �701

Aging and Survival: The Genetics of Life Span Extensionby Dietary RestrictionWilliam Mair and Andrew Dillin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �727

Cellular Defenses against Superoxide and Hydrogen PeroxideJames A. Imlay � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �755

Toward a Control Theory Analysis of AgingMichael P. Murphy and Linda Partridge � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �777

Indexes

Cumulative Index of Contributing Authors, Volumes 73–77 � � � � � � � � � � � � � � � � � � � � � � � �799

Cumulative Index of Chapter Titles, Volumes 73–77 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �803

Errata

An online log of corrections to Annual Review of Biochemistry articles may be foundat http://biochem.annualreviews.org/errata.shtml

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