Molecular Cell

Article

Spontaneous Intersubunit Rotationin Single Ribosomes

Peter V. Cornish,

1,4

Dmitri N. Ermolenko,

3,4

Harry F. Noller,

*and Taekjip Ha

1,2,

Department of Physics

Howard Hughes Medical InstituteUniversity of Illinois, 1110 West Green Street, Urbana, IL 61801, USA

Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA,University of California, Santa Cruz, Santa Cruz, CA 95064, USA

These authors contributed equally to this work*Correspondence:harry@nuvolari.ucsc.edu(H.F.N.),tjha@uiuc.edu(T.H.) DOI 10.1016/j.molcel.2008.05.004

SUMMARY

During the elongation cycle, tRNA and mRNA un-dergo coupled translocation through the ribosomecatalyzed by elongationfactorG(EF-G).Cryo-EMre-constructions of certain EF-G-containing complexesled to the proposal that the mechanism of trans-location involves rotational movement between thetwo ribosomal subunits. Here, using single-moleculeFRET, we observe that pretranslocation ribosomesundergo spontaneous intersubunit rotational move-ment in the absence of EF-G, ﬂuctuating betweentwo conformations corresponding to the classicalandhybridstatesofthetranslocationalcycle.Incon-trast,posttranslocationribosomesareﬁxedpredom-inantly in the classical, nonrotated state. Movementof the acceptor stem of deacylated tRNA into the50S E site and EF-G binding to the ribosome bothcontributetostabilizationoftherotated,hybridstate.Furthermore, the acylation state of P site tRNA hasa dramatic effect on the frequency of intersubunitrotation. Our results provide direct evidence thatthe intersubunit rotation that underlies ribosomaltranslocation is thermally driven.

INTRODUCTION

Protein synthesis is a dynamic process carried out by the ribo-some,anRNA-basedmolecularmachine.Duringproteinsynthe-sis, tRNA and mRNA are translocated through the ribosome in aseries of complex, large-scale molecular movements catalyzedby elongation factor G (EF-G) and GTP. However, translocationcan occur, albeit very slowly, in the absence of EF-G and GTP( Cukras et al., 2003; Fredrick and Noller, 2003; Gavrilova et al.,1976;GavrilovaandSpirin,1971;Pestka,1969 ).Thus,transloca-tion is a property of the ribosome itself, rather than of EF-G, andis thermodynamically favored even in the absence of GTPhydrolysis.Chemical probing studies provided the first direct evidencethat translocation takes place in two steps involving an interme-diate hybrid state ( Moazed and Noller, 1989b ). In the first step,theacceptorendsofthetRNAsmoverelativetothe50Ssubunit,from their classical A/A- and P/P-binding states into hybrid A/Pand P/E states (in which the peptidyl-tRNA is bound in the 30S A site and the 50S P site and the deacylated tRNA is bound inthe 30S P site and the 50S E site;Figure 1 A). The specific affinityof the acceptor end of deacylated tRNA for the 50S E site ( Lilletal.,1986 )helpstoaccountforthethermodynamicdrivingforcefor spontaneous formation of the hybrid state. In the secondstep, which strongly depends on participation of EF-G, theiranticodon ends move on the 30S subunit, coupled with mRNA movement, into the posttranslocational P/P and E/E states.Cryo-EM studies have identified a conformation of the ribo-some in which the 30S subunit is rotated by about 3



–10



cou-nterclockwise relative to the 50S subunit in complexes contain-ing EF-G

GDPNP (a nonhydrolyzable analog of GTP) orEF-G

GDP

fusidic acid ( Frank and Agrawal, 2000; Gao et al.,2003; Valle et al., 2003 ). This finding led to the proposal of a ratchet-like mechanism, in which translocation of tRNA andmRNA is linked to intersubunit rotational movement ( Frank and Agrawal, 2000; Frank et al., 2007; Tama et al., 2003; Valleet al., 2003 ). Recently, this model has been directly tested byformation of a disulfide bridge between ribosomal proteins S6and L2 designed to restrict intersubunit movement, resulting ina specific block in translocation ( Horan and Noller, 2007 ).The hybrid-state and ratchet models have now converged.RecentbulkFRETmeasurementscombinedwithchemicalprob-ing experiments show that the EF-G-induced rotation of the 30Ssubunit observed in cryo-EM reconstructions corresponds toformation of the hybrid state characterized by chemical probingstudies ( Ermolenko et al., 2007a, 2007b ). Although EF-G bindingwas found to stabilize the rotated, hybrid state ( Spiegel et al.,2007 ), rotation of the 30S subunit was also observed in the ab-sence of EF-G under conditions favoring the hybrid state ( Ermo-lenko etal., 2007a ), consistent withprevious biochemical exper-iments with pretranslocation complexes ( Sharma et al., 2004 ).Furthermore, spontaneous movement of two fluorescentlylabeled tRNAs relative to each other, interpreted as movementof the tRNAs between the classical and hybrid states, was ob-served in individual pretranslocation ribosomes using single-molecule FRET (smFRET) ( Blanchard et al., 2004b; Kim et al.,2007; Munro et al., 2007 ).

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Molecular Cell

, 578–588, June 6, 2008

2008 Elsevier Inc.

Although the above-mentioned evidence points to the role of ribosome structural dynamics in translocation, the underlyingmolecular mechanism of this process remains elusive. Intersu-bunit movements inferred from cryo-EM and static bulk FRETexperiments have been performed at equilibrium and on theensemble level and have yet to be observed in real time; more-over, there is so far no thermodynamic and kinetic descriptionofribosomalintersubunitmovement.Finally,theproposal,basedoncryo-EM( FrankandAgrawal,2000;Gaoetal.,2004 )andFRETstudies( Munroetal.,2007;Panetal.,2007 ),thatribosomal sub-units may occupy more than one intermediate conformationalstatehasyettobeestablished.Here,weaddressthesequestionsdirectlyusingsmFRET( Haetal.,1996 )andtotalinternalreﬂectionmicroscopy ( Zhuang et al., 2000 ). This method has been usedpreviously to probe tRNA dynamics during and after tRNA ac-commodation ( Blanchard et al., 2004a, 2004b; Gonzalez et al.,2007; Kim et al., 2007; Lee et al., 2007; Munro et al., 2007 ) andEF-Gdynamics( Wangetal.,2007 )ontheribosome.Inourexper-iments, using ﬂuorescently labeled ribosomal subunits, we usethis approach to directly monitor the dynamics of the ribosomeitself. We observe the hypothesized ratchet-like motions of indi-vidual ribosomes and characterize the determining factors of theirdynamics.Theabilityofribosomestoundergospontaneousintersubunit rotation in the absence of EF-G or GTP has strongimplications for the molecular mechanism of translocation.

RESULTSIntersubunit Movement in IndividualPretranslocation Ribosomes

We conjugated fluorescent labels to specific cysteine residuesintroduced by directed mutagenesis into ribosomal proteins S6(D41C), S11 (E75C), and L9 (N11C) ( Figure 1B) ( Ermolenko et al., 2007a, 2007b ). Proteins S6 or S11 labeled with acceptor(Cy5) dye and protein L9 labeled with donor (Cy3) dye wereincorporated into 30S and 50S subunits, respectively, by in vitroreconstitution as described previously ( Ermolenko et al., 2007a ).The labeled subunits were then associated to create two kindsof doubly labeled 70S ribosomes, 70S:S6(Cy5)/L9(Cy3) and70S:S11(Cy5)/L9(Cy3). In vitro assays showed that at least50%–60% of purified reconstituted, labeled ribosomes wereactive in in vitro translocation ( Ermolenko et al., 2007a ) and80%–100%activeininvitrotranslationofadefinedmRNA(L.Lan-caster, personal communication). In order to study the intrinsicstructural dynamics of the pretranslocation ribosome, the pep-tidyl-tRNA analog N-Ac-Phe-tRNA

Phe

was bound to the A site of ribosomes containing deacylated tRNA

fMet

bound to the P sitein the presence of a defined mRNA. Pretranslocation ribosomecomplexes were then immobilized in polymer-passivated micro-scope slide/coverslip chambers via a biotin-derivatized DNA oligonucleotide annealed to the mRNA ( Figure 1C) ( Blanchard et al., 2004b ) and were visualized using total internal reflectionmicroscopy ( Zhuang et al., 2000 ). This immobilization approachpreserved the ribosome’s translocation activity (see below).Time traces of individual S6-Cy5/L9-Cy3 pretranslocationcomplexes showed spontaneous, time-dependent anticorre-lated changes in donor (Cy3) and acceptor (Cy5) fluorescenceintensities ( Figure 2 A). Calculation of apparent FRET efficiency(FRET = I

Cy5

/[I

Cy5

+ I

Cy3

]) from donor (I

Cy3

) and acceptor (I

Cy5

)intensities revealed that pretranslocation ribosomes ﬂuctuatebetween high (0.56) and low (0.40) FRET states. smFRET mea-surements performed with the Cy3 and Cy5 dyes reversedgave similar results (data not shown). Time traces recorded forS11-Cy5/L9-Cy3 ribosomes show a similar pattern of spontane-ous ﬂuctuations but inverted from that of the S6/L9 construct(data not shown), because S11 moves closer to L9 in the hybridstate, whereas S6 moves away from L9 ( Ermolenko et al.,2007a ). Below, we present only data from the S6/L9 construct,because of the previously demonstrated strong anticorrelationbetween the FRET changes for the S6/L9 and S11/L9 dye pairs( Ermolenko et al., 2007a ).The high (0.56) FRET state for the S6/L9 pair corresponds tothe nonrotated conformation of the ribosome, in which thetRNAs are bound in the classical state, whereas the low (0.4)FRET state corresponds to the conformation in which thesmall ribosomal subunit is rotated and the tRNAs are bound in

Figure 1. Experimental Design

(A) Cartoon showing the movement of deacylatedtRNA

fMet

(initially in the P site) and peptidyl-tRNA analogN-Ac-Phe-tRNA

Phe

(initiallyinAsite)duringtranslocation between classical pretranslocation,hybridpretranslocation,andclassicalposttranslo-cation states.(B) Positions of ﬂuorescent dyes (orange spheres)coupled to proteins L9, S6, and S11 in the 70S ri-bosome, viewed from the E site interface side of the crystal structure ( Korostelev et al., 2006 ). The50S subunit is on the left (23S and 5S rRNAs arein gray, proteins in magenta), and the 30S subunitis on the right (16S rRNA in cyan, proteins in blue).The E site tRNA (red) can be seen spanning theinterface. The red arrows indicate the direction of intersubunit rotation accompanying hybrid-stateformation.(C) Ribosomes were immobilized by hybridizationof the 3

tail of the mRNA to a biotin-derivatizedDNA strand that was bound via neutravidin toa quartz coverslip.

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Spontaneous Movement in Single Ribosomes

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, 578–588, June 6, 2008

2008 Elsevier Inc.

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the A/P and P/E hybrid states ( Ermolenko et al., 2007a, 2007b ).Thelatterconformationisequivalenttothe‘‘ratchetedstate’’ob-served in cryo-EM studies of complexes of the ribosome boundwithEF-G( FrankandAgrawal,2000;Valleetal.,2003 ).Thus,oursingle-ribosome traces show that the pretranslocation ribo-some, in the absence of EF-G or GTP, ﬂuctuates spontaneouslybetween the rotated and nonrotated conformations, corre-sponding to the hybrid and classical states, respectively.To askwhether the ribosomal subunits also move through anyadditional, previously unobserved transient rotational states, thepresenceofwhichcouldbemaskedbynoiseinourFRETtraces,we subjected our data to a hidden Markov modeling (HMM)algorithm ( McKinney et al., 2006 ). This approach allows fordetermination of the number of states present in the systemand the rates of exchange between them. HMM analysis of theS6-Cy5/L9-Cy3 pre-translocation complex (612 total moleculesshowingonaverage30transitionspermolecule;Table1 )assum-ingthreestates(Figures2Cand3 )showedthatthepretransloca- tion complex ﬂuctuates between just two distinct states, nonro-tated and rotated, with a forward rotation (nonrotated to rotated)rate of 0.27 ± 0.08 s

and a reverse rotation (rotated to nonro-tated)rateof0.19±0.02s

at25



Cunderourinvitroconditions( Figures 2B and 2C;Table 1 ). ThesameanalysisperformedonribosomescontainingonlyaPsitetRNA(tRNA

fMet

ortRNA

Met

)alsoresultedinjusttwoobservedFRET states ( Table 1and seeFigure S1available with this article online). In addition, dwell-time analysis for all three complexesﬁt to a single exponential decay, consistent with two states( Figure S2 ).

Figure 2. HMM Analysis of FRET DataObtained from S6-Cy5/L9-Cy3 Pretrans-location Ribosomes

(A) Representative trace showing ﬂuorescenceintensities observed for the Cy3 donor (green)attached to L9 and a Cy5 acceptor attached toS6 (red) in ribosomes containing tRNA

fMet

in theP site and N-Ac-Phe-tRNA

Phe

in the A site.(B) Schematic showing the observed FRET valuesfor the two states and the forward and reversetransition frequencies (k

and k

).(C) Transition density plot (TDP) for the pretranslo-cation complex. The TDP is constructed by plot-ting values for each transition based upon theFRET value from which the transition originated(x axis) and to which FRET value the transitionends (y axis). The transition paths are indicatedby the broken red arrows.

Effects of the Acylation Stateof the P Site tRNA and EF-Gon Intersubunit Rotation

We next asked how the acylation state of P site tRNA and EF-G binding affects thenonrotated/rotated states’ equilibrium.The equilibrium constant,

rotated

non

rotated



, isdetermined from double Gaussian ﬁts to smFRET histograms( Dahan et al., 1999 ) built from several hundred molecules foreach construct ( Figure 4andFigure S3;Table 2 ). A majority (75%) of posttranslocation ribosomes, containing the peptidyl-tRNAanalogN-Ac-Phe-tRNA

Phe

boundinthePsitewithavacant A site, exhibited a high FRET value ( Figure 4 A), corresponding totheclassical,nonrotatedconformation,inagreementwithchem-ical probing ( Moazed and Noller, 1989b ) and bulk FRET ( Ermo- lenkoetal.,2007a )experiments.Inacomplexcontainingadiffer-ent peptidyl tRNA, fMet-tRNA

fMet

, bound to the P site, 66% of ribosomeswere alsointhenonrotated conformation ( Figure4B).Likewise, 52% and 79% of authentic posttranslocation com-plexes obtained by incubation of the pretranslocation complex(the former containing the N-Ac-Phe-tRNA

Phe

bound to the A site and deacylated tRNA

fMet

bound to the P site and the lattercontaining N-Ac-Phe-tRNA

Phe

bound to the A site and deacy-lated tRNA

Tyr

bound to the P site) with EF-G

GTP were foundin the nonrotated conformation ( Figures 4C and 4D). Sixty-onepercent of vacant ribosomes (i.e., ones lacking tRNA) alsoexhibited a high FRET value ( Figure 4K).In contrast to posttranslocation complexes, ribosomes con-taining deacylated tRNA

fMet

in the P site without an A site tRNA showed a majority of ribosomes (77%) in the low-FRET rotatedstate( Figure4F).AnevenmorepronouncedeffectwasobservedinthecaseofdeacylatedtRNA

Phe

,whichshifted85%oftheribo-some population to the rotated state ( Figure 4E), consistent withthe higher propensity of tRNA

Phe

to occupy the hybrid P/E state,compared with tRNA

fMet

( Dorner et al., 2006; Spiegel et al.,2007 ). A similar difference was also observed between tRNA

fMet

and tRNA

Met

( Studer et al., 2003 ). In contrast, only 29% of ribo-somes containing only a tRNA anticodon stem loop (ASL) boundto the P site were found in the hybrid, rotated state ( Figure 4L),

Molecular Cell

Spontaneous Movement in Single Ribosomes

580

Molecular Cell

, 578–588, June 6, 2008

2008 Elsevier Inc.