Millisecond dynamics of RNA polymerase IItranslocation at atomic resolutionDaniel-Adriano Silvaa, Dahlia R. Weissb, Fátima Pardo Avilaa, Lin-Tai Daa, Michael Levittc, Dong Wangd,1,and Xuhui Huanga,1

aDepartment of Chemistry, The Hong Kong University of Science and Technology, Kowloon, Hong Kong; bDepartment of Pharmaceutical Chemistry,University of California, San Francisco, CA 94158; cDepartment of Structural Biology, Stanford University, Stanford, CA 94085; and dSkaggs School of Pharmacyand Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093

Edited by Robert Landick, University of Wisconsin-Madison, Madison, WI, and accepted by the Editorial Board March 27, 2014 (received for reviewAugust 21, 2013)

Transcription is a central step in gene expression, in which theDNA template is processively read by RNA polymerase II (Pol II),synthesizing a complementary messenger RNA transcript. At eachcycle, Pol II moves exactly one register along the DNA, a processknown as translocation. Although X-ray crystal structures havegreatly enhanced our understanding of the transcription process,the underlying molecular mechanisms of translocation remainunclear. Here we use sophisticated simulation techniques toobserve Pol II translocation on a millisecond timescale and at at-omistic resolution. We observe multiple cycles of forward andbackward translocation and identify two previously unidentifiedintermediate states. We show that the bridge helix (BH) playsa key role accelerating the translocation of both the RNA:DNAhybrid and transition nucleotide by directly interacting with them.The conserved BH residues, Thr831 and Tyr836, mediate theseinteractions. To date, this study delivers the most detailed pictureof the mechanism of Pol II translocation at atomic level.

Markov state model | molecular dynamics | trigger loop

The RNA polymerase is the central component of gene ex-pression in all living organisms, transferring genetic infor-

mation from DNA to RNA. In eukaryotes, the RNA polymeraseII (Pol II) enzyme is responsible for transcribing DNA intomessenger RNA. In the past decade, a number of X-ray crys-tallographic structures of Pol II have been obtained at differentstages of the transcription process, providing a static pictureof how this complex machine performs its function (1, 2).Transcription is a multistep process consisting of initiation,elongation, and termination, where elongation is composed ofconsecutive nucleotide addition cycles (NACs). In each NAC,the NTP substrate first diffuses into Pol II active site throughthe secondary channel (3–5) or alternatively the main channel(6). Upon correct NTP binding to the Pol II active site, the triggerloop (TL) conformation switches from an inactive open state toan active closed state (7). The closure of the active site subse-quently facilitates the catalysis of the nucleotide addition re-action (7), followed by release of the pyrophosphate ion (PPi). Toproceed to the next NAC, Pol II must translocate from a pre-translocation state, in which the active site is still occupied by thenewly added nucleotide at 3′-RNA, to a posttranslocation state.During translocation, the template DNA and RNA must moveby exactly one register, once again creating a free insertion site(i site) (1, 2, 5, 8–13).Although static snapshots of X-ray structures of Pol II pre-

translocation and posttranslocation states are valuable, the dy-namics underlying the fundamental RNA polymerase translocationmechanism remain poorly understood (14). Two models of trans-location have been proposed based on structural, biochemical, andgenetic approaches. On one hand, for single subunit T7 RNAP,PPi release is suggested to be mechanically coupled to the openingmotion of the O-helix (counterpart of TL) and subsequenttranslocation, referred to as the “power-stroke” model (15, 16).On the other hand, the Brownian ratchet model is proposed for

translocation in multisubunit RNA polymerases (14, 17–19). Inthis model, the system can rapidly interconvert between the pre-translocation and posttranslocation states without the requirementof NTP hydrolysis. The motion is facilitated by thermal oscillation ofthe bridge helix (BH) between the straight and bent conformations;the binding of the incoming NTP will then stabilize forward trans-location (14, 17–19). However, the dynamics at the atomic level andthe details of the mechanism of Pol II translocation remain obscureand direct experimental approaches are limited.Molecular dynamics (MD) simulations can provide dynamic

information at atomic resolution and thus complement experi-mental approaches to elucidate the mechanisms of Pol IItranslocation. Indeed, previous all-atom MD simulation studieshave provided valuable insights into the dynamics of Pol II (9,20–22). However, it is important to point out that previous MDsimulations are limited to a few hundred nanoseconds, at whichproteins may only undergo local conformational changes such asside-chain rotations and loop motions. These MD simulationsfall far short of biologically relevant timescales of Pol II trans-location (tens of microseconds to millisecond or even longer)(23, 24). Directly simulating the millisecond timescale of a hugesystem like Pol II in explicit solvent (nearly half a million atoms)is not yet possible, even with modern specialized simulationhardware (25–27). Therefore, a major challenge for simulatingPol II translocation is to reach the biologically relevant timescalefor this complex cellular machinery.

Significance

In this study, by using molecular dynamics simulations andMarkov state models, we reveal that RNA polymerase IItranslocation is driven purely by thermal energy and does notrequire the input of any additional chemical energy. Our simu-lations show an important role for the bridge helix: Large thermaloscillations of this structural element facilitate the translocationby specific interactions that lower the free-energy barriers be-tween four metastable states. Among these states, we identifytwo previously unidentified intermediates that have not beenpreviously captured by crystallography. The dynamic view oftranslocation presented in our study represents a substantialadvance over the current understanding based on the staticsnapshots provided by X-ray structures of transcribing complexes.

Author contributions: D.-A.S., D.R.W., D.W., and X.H. designed research; D.-A.S., D.R.W.,F.P.A., L.-T.D., and M.L. performed research; D.-A.S., M.L., and X.H. contributed newreagents/analytic tools; D.-A.S., F.P.A., L.-T.D., and M.L. analyzed data; and D.-A.S., D.R.W.,F.P.A., M.L., D.W., and X.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.L. is a guest editor invited by the Editorial Board.

Freely available online through the PNAS open access option.

See Commentary on page 7507.1To whom correspondence may be addressed. E-mail: xuhuihuang@ust.hk or dongwang@ucsd.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1315751111/-/DCSupplemental.

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Recently, Markov state models (MSMs) built from manysimulations (each as short as a few nanoseconds) have beenapplied to study protein folding and function at microsecond oreven millisecond timescales (28–35). To overcome the timescalegap, we have seeded MD simulations along low-energy trans-location pathways predicted by the Climber algorithm (36), andthen we have used MD simulations to construct a MSM (Fig.S1). This strategy allows us to investigate the dynamics of the PolII translocation at atomic resolution and millisecond timescales.Here we report the complete Pol II translocation event at atomiclevel. We found that Pol II can oscillate between the pre-translocation and posttranslocation states with an averagetimescale of tens of microseconds for each transition. Our sim-ulation results are in general support of the Brownian ratchetmechanism, although the mechanical analogies of reciprocatingand stationary pawls may not precisely describe a system drivenby stochastic thermal motion such as Pol II. Instead, we see theBH playing a central role in facilitating the translocation of theRNA:DNA hybrid and that of the transition nucleotide (TN) byreducing free-energy barriers. This provides novel insights intothe metastable intermediate states, the rate-limiting step, and thedriving force of the Pol II translocation process, which allows usto propose a detailed mechanism for Pol II translocation.

ResultsHigh-resolution structures of the Pol II complexes have greatlyenhanced our understanding of the transcription mechanism;however, crystal structures can only capture static snapshots ofthe starting and ending points of translocation: pretranslocationand posttranslocation states. To investigate the detailed molec-ular events underlying Pol II translocation, we constructeda MSM from all-atom MD simulations (Material and Methods).

Simulations Reveal Spontaneous Millisecond Pol II Translocation.Using our MSM, we have generated a synthetic trajectory re-vealing the millisecond dynamics of the large system of Pol IItranscription complex in explicit solvent (∼426,000 atoms in to-tal; Fig. S2), providing an observation of full cycles of Pol IItranslocation events at the atomic level. We observed that Pol IIoscillates repeatedly between the pretranslocation and post-translocation states within a millisecond in the absence of theincoming NTP (Fig. 1A). The average translocation timescale isaround tens of microseconds. A movie for a 7-μs segmentexhibiting a translocation event is available as Movie S1.

Four-State Asynchronous Translocation.Our simulations reveal thatthere are four metastable states along the Pol II translocationpathway, two are already known and two are previously un-identified metastable states (structure coordinates for these twostates are available in Supporting Information). In addition to pre-translocation and posttranslocation states previously captured byX-ray crystallography (states 1 and 4; Figs. 1 B and E), we identifiedtwo previously unidentified metastable intermediate states (states 2and 3; cyan and orange dashed lines in Fig. 1 A, C, and D). In thefirst intermediate state (state 2, Dataset S1), the backbone of theupstream DNA and RNA has been translocated, whereas the baseof the TN at the DNA i+2 position along the DNA lags behind,staying directly above the BH to form stacking interactions with theBH residue Tyr836 (Fig. 1C). Furthermore, the interaction betweenThr831 and the DNA nucleotide (i+1 position) as found in state 1 islost in state 2 due to the slight rotation of the BH. This loss ofinteraction in state 2 is compensated by the newly formed stackinginteractions between the TN base and Tyr836, which lower theenergetic barriers for translocation. In the second intermediatestate (state 3, Dataset S2), the interactions between Thr831 and theDNA nucleotide are reestablished with the TN base, which hascrossed the BH and reached a position only a few angstroms awayfrom the canonical i+1 position. The position of the TN in state 3 ispoised to form some initial/partial interactions with the incomingNTP, if it is available. To complete the full translocation, the TN

further moves into the canonical i+1 position that allows Watson–Crick base pairing to the incoming NTP (state 4).We found that the TN moves asynchronously from the rest of

the upstream RNA and DNA in the hybrid region. As shown inFig. 2A, the upstream RNA:DNA hybrid translocates simulta-neously via a single-step transition from state 1 to reach theirfinal register positions at state 2. They then remain in the sameposition throughout state 2 to state 4 (Fig. 2B). Accordingly, themagnesium ion (Mg2+A) also switches its binding nucleotide,from the i+1 to i−1 RNA nucleotide during the transition fromstate 1 to state 2 (Fig. S3A). In sharp contrast, the TN base lagsbehind the translocation of the upstream RNA:DNA hybrid.

Fig. 1. (A) RMSD of the active site with respect to the posttranslocationstate as a function of time for the 1-ms MSM simulation trajectory (Materialsand Methods). The segment between 10 and 35 μs is shown in the insert. Theaverage RMSD for states 1–4 is shown by four dashed lines colored black,cyan, orange, and green for states 1–4, respectively. (B–E) Representativeconformations of the metastable states identified by our model. Detailedviews of the active site are shown for each of the four states identified byour model. Nucleic acids, ions, Thr831, and Tyr836 are shown in a sphererepresentation, whereas protein elements are shown in cartoon represen-tation. The RNA (red), template DNA strand (cyan), BH (green), TL (purple),Mg2+ A (magenta), Rpb1 Thr831 (wheat), Rpb1 Tyr836 (yellow), and TN(orange) are shown. (B) State 1 (S1) corresponds to the pretranslocationstate, analogous to the one found in X-ray crystallographic studies. (C and D)States 2 and 3 are intermediates of the translocation identified by the MSM.(E) State 4 also corresponds to the posttranslocation state found in X-raycrystallographic studies.

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Specifically, the TN base translocates through a three-stepmechanism. In the first step (state 1 to state 2), the TN basemoves from the pretranslocation position to the top of the BH,halfway to the position of posttranslocation state, maintainingstacking with BH Tyr836 residue (Figs. 1C and 2B). Next (state 2to state 3), the TN base crosses over the BH but is still a fewangstroms away from the canonical posttranslocation state. Fi-nally (state 3 to state 4), the transition base rotates to the ca-nonical position of posttranslocation state, which allows full basepairing with the incoming NTP. The intermediate state’s identityis consistent with previous translocation intermediate structurestrapped either by α-amanitin or DNA damage (13, 37), in whichthe TN base is located on top of the BH.

The Thermal Oscillation of the BH Drives Translocation. In our sim-ulations, we observed thermal oscillation of the BH between thebent and straight conformations throughout states 1–3 (Fig. S4),whereas state 4 did not exhibit significant bending motion. Thisthermal bending of the BH is tightly correlated to translocationof the upstream DNA:RNA hybrid backbone (states 1–3). Thebent BH occupies part of the active site interacting with theDNA nucleotide at the i+1 position and facilitating the motionof upstream template DNA to the next register (Fig. 3A and Fig.S5B). In particular, BH residues 831–836 are involved in thisinteraction with the DNA template. A correlation analysis con-firms that the motion of this segment is highly coupled with theDNA i+1 nucleotide in state 1 (Fig. 3C). Thr831 is within thissegment, consistent with the observation that Thr831 is in directcontact with the DNA i+1 nucleotide as in the pretranslocationcrystal state (5). In addition, the thermal fluctuation of the BHmay also help the translocation of the TN base. The TN base isstabilized by stacking interactions with Tyr836 during the transitionfrom state 1 to state 2 (Fig. 4A). Its motion is also highly correlatedwith the BH residues 831–836 when the TN base crosses over theBH (states 2 and 3) (Fig. 3C).

We further analyzed the bending features of the BH at theresidue level. We found that the direction of BH bending is to-ward the active site (Fig. 3A and Fig. S5B). Among all of theresidues in BH, the central segment (residues 827–831; Fig. 3B)undergoes the largest bending motion (with a magnitude of ∼8Å) (Fig. 3A). The intrinsic bending motion of the BH may beenabled by two adjacent highly flexible glycine residues (Gly819and Gly820). This Gly–Gly pair is referred to as a “hinge,” andwe found that it can transiently lose its α-helix secondary struc-ture (Fig. S5C), consistent with previous MD simulation studieson a short timescale (21). We also examined if the BH bending iscoupled with the clamp motion by computing its cross-correla-tion (Fig. S6). The results suggest that the translocation maynot be strongly coupled to the clamp opening during theelongation phase.Our findings are consistent with the Brownian ratchet mech-

anism of translocation (17) and provide atomic details for thismodel. The bent BH facilitates the upstream translocation ofRNA:DNA hybrid. In the next step, the TN is translocated overthe BH, again facilitated by interactions with the fluctuation of theBH. The translocation motion, which takes place on a timescale oftens of microseconds, is reversible until the incoming NTP sta-bilizes the system in the posttranslocation state.

Fig. 2. Translocation of the RNA:DNA hybrid is not synchronous with thetranslocation of TN. (A) A cartoon of the four metastable states, S1 to S4,shows the order of backbone and nucleotide translocation. From S1 to S2the backbones of the RNA:DNA hybrid (red and cyan) translocate frompretranslocation to posttranslocation positions, whereas the TN (orange)lags behind and stacks with the BH (green). From S2 to S3 the TN crosses overthe BH toward the active site but still remains stacked with the BH. From S3to S4 the TN moves into the active site, losing the stacking with the BHand completing the translocation. (B) The plots show the distance to positionin posttranslocation state for the backbone phosphate versus distance toposition in the posttranslocation state for the nucleotide base. For DNA(Top) and RNA (Middle) the values are averaged over the eight upstreamnucleotides of the DNA and RNA (also see Fig. S3), respectively. For the DNATN (Bottom) there is just one phosphate and one base, so no averagingis needed.

Fig. 3. The dynamics of the BH and TL direct the translocation. (A) The BHmotion projected along the alpha helix axis shows that the bending can beas large as 10 Å, with a preference to bend toward the upstream RNA:DNAhybrid (see Fig. S5 for details of the projection). The cyan and orange dashedovals illustrate the i+1 and i+2 template DNA positions. (B) The RMS fluc-tuation values of the Cα atoms in the BH show that its maximum bendingoccurs in the middle of the helix. (C) In the pretranslocation state (S1) thehelix can bend enough to interact with the base in the i+1 position, and thecorrelation plots show that indeed the movement of the helix is correlatedto the displacement of the i+1 DNA position (blue). However, in bothintermediates the TN motion is tightly correlated with the BH. The first in-termediate (S2) centered on the residues next to the Tyr836 and the secondintermediate (S3) centered with the residues surrounding the Thr831. Fi-nally, in the posttranslocation (S4), most of the correlation is lost. (D) Thegraph of the cross-correlation between the TL and the BH Cα atoms (Left)shows that the motion of the TL in these segments is highly correlated to themotion of the BH.

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The Motions of TL and BH Are Tightly Coupled. Our simulationsreveal that the motions of the TL and BH are tightly coupledduring the translocation process. In particular, BH residues831–836 couple with the TL through two segments: residues1076–1082 and 1097–1103 (Fig. 3D). We observed two mainconformations of the TL when the BH is bent: one involves theLeu1081 in the wedged state (37), whereas in the other, Leu1081is not wedged (Fig. S7), indicating that the bent BH and wedgedLeu1081 do not always coincide in the dynamic ensemble. Anadditional TL segment (1097–1103) far away from the active sitemay also affect the dynamics of translocation because motion ofthat segment is correlated to BH motion (Fig. 3C). Previousexperiments show that mutations on these segments affect the invitro elongation rate. The elongation rate is increased by thesingle mutations G1097D, L1101S, and E1103G, whereas themutation N1082S decreases the elongation rate. Furthermore,the mutations Q1078 ⇨ A/E/N, L1081 ⇨ A/I/F/G, and N1082Agive rise to lethal phenotypes (38, 39). We suggest that the TLmay play a role in translocation by enhancing the thermal fluc-tuation of the BH through the highly coupled motion betweenthese two structural motifs (6, 7, 37–39).

The Free-Energy Landscape Suggests a Single Rate Limiting Step inTranslocation. We calculated the free-energy landscape of thetranslocation using the MSM (Fig. 5) and identified a singlehigh free-energy barrier separating the pretranslocation (state1) and first intermediate (state 2) states. Therefore, this tran-sition is the rate-limiting step of Pol II translocation and occurson a timescale of tens of microseconds. This rate-limitingtransition is characterized by the backbone translocation of theupstream RNA:DNA hybrid (Fig. 2) and a stabilizing stackinginteraction between the BH residue Tyr836 and the TN (Figs.1C and 4A). There is no significant free-energy barrier fortransitions among states 2, 3, and 4, which are all located ina wide flat free-energy basin.A π–π stacking interaction in state 2 between the TN base and

the Tyr836 side chain helps the system overcome the rate lim-iting free-energy barrier between states 1 and 2. This interactioncan only be seen in simulations and is present in neither pre-translocation nor posttranslocation structures (Fig. 4A). Tyrosineat this position is highly conserved, which might be related to itsimportance in transcription (Fig. 4B). Therefore, to further ex-amine the functional importance of this stacking interaction,we ran single mutant simulations at this position. We selectedthe initial conformations from around the maximum height ofthe free-energy barrier between states 1 and 2 (i.e., around the

transition state). Theoretically, half the simulations shouldtranslocate forward, whereas the other half will translocatebackward. Indeed, in the WT simulation, around half the con-formations move toward the posttranslocation state. The phe-nylalanine mutant (Y836F) can form π–π stacking interactionswith the transition base and therefore behaves similarly to thewild-type simulations. However, the valine mutant (Y836V)cannot form stacking interactions, and simulations are morelikely to return to the pretranslocation state (Fig. 4C). Theseresults are consistent with mutagenesis experiments on a relatedmultisubunit RNAP (40), where valine mutants at this positionhave lower transcription activity compared with phenylalanineand WT (with tyrosine).

DiscussionWe have simulated the molecular dynamics of complete Pol IItranslocation at atomistic resolution and millisecond timescalesusing MSMs. Notably, our simulations mimic the translocationprocess after the PPi release but before the loading of the nextNTP and with the TL in an open conformation. Under theseconditions, we observe a number of reversible transitions be-tween pretranslocation and posttranslocation states within a milli-second. Our choice of the TL in an open conformation is based onrecent fluorescence experimental observations (23) and compu-tational studies (20), both suggesting that the opening of the TL isa prerequisite for full translocation.Our results reveal that Pol II translocation is driven purely by

thermal energy and does not require the input of any additionalchemical energy. The thermal fluctuations of the BH betweenbent and straight conformations facilitate the translocation ofthe upstream RNA:DNA hybrid through the direct contactbetween the BH and i+1 base pair of the upstream RNA:DNAhybrid. This is the rate-limiting step of translocation. Inthe next step, the TN is translocated to become part of theRNA:DNA hybrid; this translocation is also facilitated by thethermal fluctuation of the BH that interacts with the TN.The incoming NTP serves to stabilize the system in theposttranslocation state.In our model (Fig. 6), we did not observe any asymmetric step-

wise translocation for the upstream DNA and RNA as suggestedby structural studies of transcription initiation complex withshort RNA:DNA hybrid (12, 41). Instead, both the nascent RNAchain and upstream DNA translocate simultaneously in ourtranscription elongation complex. This may reflect a differenttranslocation mechanism between the early transcription initia-tion step (with an unstable short RNA:DNA hybrid) and

Fig. 4. Stacking between BH residue Tyr836 and the TN base plays an im-portant role in facilitating translocation. (A) A representative structure fromstate 2 shows π-stacking between Tyr836 (yellow) and the TN base (orange).Such stacking is not seen in the X-ray structures of Pol II either before (PDBID: 1I6H) or after translocation (PDB ID: 2E2H). (B) Multiple sequence align-ment across different species shows conservation of Tyr836, Thr831, andGly835. These same residues show highly correlated motion to the BH andthe TN. (C) Mutant simulations reveal that disrupting the π-stacking in-teraction between Tyr836 and the TN hinders forward translocation to theposttranslocation state.

Fig. 5. The schematic free-energy landscape of translocation. The pathwayfrom the pretranslocation state S1 (red curve) to the posttranslocation stateS4 (green curve) has two metastable intermediates (S2 and S3; blue andorange curves). Representative structures of the states are displayed to-gether with their equilibrium populations and the average times (in μs) fortransitioning between them. The transition from S1 to S2 is rate-limiting. Allof the major pathways connecting S1 and S4 have to go through S2 (seeSupporting Information for details of the pathway analysis).

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productive elongation steps (with a stable 8–9 nt long RNA:DNA hybrid).We have elucidated the detailed molecular mechanism of the

bending motion of the BH: unfolding of the GG segment(819–820) promotes bending of the adjacent segment (827–831),and this bending is further propagated to the DNA templatethrough a third segment (831–836). Mutations of these residuesin a related RNAP decrease the in vitro transcription rate (38),and footprinting experiments show that mutations near the GGsegment that increase the bending of the BH can promotetranslocation (42, 43). In addition, a previous structural studyrevealed that Pol II inhibitor α-amanitin forms a hydrogen bondwith E822 (44), likely trapping the BH in a straight conformationand hindering the bending motion of the BH. Tyr836 in the thirdsegment facilitates the system in overcoming the largest free-energy barrier of translocation through π–π stacking interactionswith the TN base. It is important to note that the time scale ofthe BH bending oscillation is much faster than translocation.The BH may oscillate between bent and straight conformationsmany times within a single translocation event. The bent BHconformation can be trapped in the elemental paused RNAP asrevealed by a recent structural study (39). In our model, themotion of the TL is also coupled to the BH through two seg-ments; one is in direct contact with the BH, whereas the other isfar from the active site. Mutations in these two segments willaffect dynamics of TL conformation change between open andclosed conformations, the transcription rate, and fidelity (G1097D,L1101S, or E1103G) or even result in lethal phenotypes (e.g.,Q1078 ⇨ A/E/N) (38). It is conceivable that future biochemical andstructural studies could exploit mutations of key residues in theBH and TL, as well as modifications/damage to the template

DNA strand, that alter the interactions between Pol II andDNA during the translocation. In particular, we hypothesizethat mutations or modifications involving the Tyr836 may helptrap the intermediate states of translocation.Our simulations of translocation events, together with pre-

vious simulations (9, 20–22), provide important mechanistic andkinetic insights of several key steps in Pol II transcription. Ourprevious simulation study has suggested that the PPi release israpid [at ∼1 μs (9)]. NTP loading has been suggested to occur ina few milliseconds (45), whereas the existence of a prebindingsite may further accelerate this process. In the current study,we show that translocation can occur at timescales of tens ofmicroseconds, which is relatively fast compared with the elon-gation rate (∼100 ms per base pair in vivo). However, we notethat the rates obtained from our MSM may be overestimatedunder various scenarios, e.g., when there exist off-pathway in-termediate states, which have not been sampled by the seedingMD simulations. Therefore, we suggest these rates to be treatedas the upper limit of the rates for translocation. In future studiesit will be interesting to simulate a full cycle of nucleotide addi-tion, including DNA melting and annealing, NTP loading, TLclosure/folding, catalysis, PPi release, and TL opening/unfolding.Additionally, it would be important to consider a model ofa complete transcription bubble and other aspects known toaffect the native Pol II elongation complex, such as the effectof Mg2+ and other ions. Such studies may allow us to connectstructural snapshots and lead to a complete understanding of thedynamics of Pol II transcription.

Materials and MethodsGenerating Initial Low-Energy Pathways of Translocation. We first generatedmodels of both pretranslocation and posttranslocation states based onexisting crystal structures (see Supporting Information for details of modelconstruction). We then obtained two independent pathways along thedirections of forward and backward translocation. These initial pathwayswere produced using a modified version of the Climber algorithm (36) (seeSupporting Information for details).

Seeding MD Simulations. We performed two rounds of MD simulations. Thefirst round of simulations was initiated from structures along the two low-energy pathways generated by the Climber algorithm, and its objective wasto relax the system from these initial pathway (44 × 20 ns NPT simulations at1 bar, 300 K or 310K). We then select 80 representative conformations fromthese simulations as starting points for a production round of MD simu-lations (80 × 20 ns NVT simulations at 310 K). The second rounds of simu-lations were used to build the MSM. All of the simulations were performedusing the Amber03 Force Field (46) and Groningen Machine for ChemicalSimulations 4.5 software (47). The Pol II complex was solvated in a water box,and counter ions were added to make the system neutral. Total system sizewas 426,059 atoms. Long-range electrostatic interactions were treatedusing the Particle-Mesh Ewald method. See Supporting Information forfurther details.

Constructing and Validating MSMs. To construct MSMs, we divided all MDconformations (∼80 K) into 976 microstates using the K-centers clusteringalgorithm implemented in MSMBuilder package (48) (see Supporting In-formation for further details). The implied timescale plots displayed a pla-teau after a lag time of 4 ns, indicating that the model is Markovian at thisor a longer lag time (Fig. S8E). This 976-state MSM has been further vali-dated by successfully reproducing the probability curves for the system toremain in a certain microstate directly computed from the original MDsimulations (Fig. S8F). Finally, we compared the initial Climber pathwaysagainst the MSM results by projecting them onto a set of common reactioncoordinates using the Isomap dimensionality reduction technique (49). Thisanalysis confirmed that the sampling from MSMs has diffused away signif-icantly from the initial pathways, indicating that the initial pathways do notgovern the final results of the MSMs (Fig. S9B). All of the quantitativeproperties reported in this work are computed exclusively from the 976-state MSM.

Generating Millisecond Trajectories from MSMs. We have sampled the tran-sition probability matrix of our MSM with a lag time of 5 ns to produce themillisecond MSM simulation trajectory.

Fig. 6. A model of Pol II translocation. At the pretranslocation state (S1),the oscillation of the BH is large enough to interact with the i+1 DNA nu-cleotide, which can then facilitate the motion of the RNA:DNA hybrid to-ward the posttranslocation state. At the first intermediate state (S2), thebackbone of the upstream RNA:DNA hybrid has been translocated, whereasthe TN still lags behind, stabilized by a stacking interaction with Tyr836. Theactive site is empty, which may permit entrance of the incoming NTP to the isite. At the second intermediate state (S3), the continuous oscillation of theBH further facilitates TN crossing over it, while maintaining strong inter-actions, mainly through residue Thr831. The position of the TN in S3 mayalready allow partial interaction with the incoming NTP. In the final steps,the TN moves to its final i+1 posttranslocation position (S4). The incomingNTP may then lock the system in the posttranslocation state by entering tothe i site. The transparent surface surrounding the BH represents its overalldisplacement in each state. The BH residues that have correlated motionwith the i+1 DNA nucleotide, TN, and both of them (i+1 DNA nucleotide andTN) are displayed in blue, green, and turquoise, respectively. The thickness ofarrows that connect different states is proportional to the rate of thetransitions between them.

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Clustering Microstates into Four Metastable States. To visualize the trans-location mechanisms, we have clustered the microstates into four metastablestates using the Perron cluster cluster analysis algorithm in the MSMBuilderpackage (48).

Mutant Simulations. Using a conformation that is near to the transition statebetween the pretranslocation (S1) and the first-intermediate (S2) as the initialpoint, we generated the in silico mutants of Rpb1: Y836F and Y836V. Five 20-ns MD simulations (NVT, T = 310 K) were performed for each of the mutantsand the wild-type protein.

Cross-Correlations. To determine whether pairs of elements (e.g., a residueand a nucleotide) have concerted dynamics we have calculated the Pearsoncorrelation coefficient of the covariance matrix.

Measurement of BH Bending. The tips of the BH (Rpb1, residues 811–815 and841–844) were aligned to the structure of an idealized α-helix. We then

measured two reaction coordinates: (i) the distance of a vector to the centerof the idealized α-helix and (ii) the angle between the previous vector anda reference vector (for convenience, we defined that in an idealized α-helixthe angle of the Tyr836 is 90°).

ACKNOWLEDGMENTS. The authors would like to thank Fu Kit Sheong foruseful discussions. We acknowledge the Hong Kong Research Grant Council(Grants 661011, AoE/M-09/12, M-HKUST601/13, and T13-607/12R), NationalBasic Research Program of China (973 Program 2013CB834703), and NationalScience Foundation of China (Grant 21273188) (to X.H.); National Instituteof General Medical Sciences Grant F32GM093580 and National Institutesof Health (NIH) Grant U54 GM072970 (to D.R.W.); NIH Grants GM085136and GM102362, Sidney Kimmel Foundation for Cancer Research GrantSKF-12-014, and University of California, San Diego, Startup fund (to D.W.);Hong Kong PhD Fellowship and Consejo Nacional de Ciencia y Technología Fellow-ship 215482 (to F.P.A.); and NIH Grant GM063817 (to M.L., who is the Robert W.and Vivian K. Cahill Professor of Cancer Research). Computing resources wereprovided by the National Supercomputing Center in Shenzhen and Aitzaloa clus-ter in Universidad Autónoma Metropolitana of Mexico City.

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