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Mini Review: A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0

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RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine
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  (This is a sample cover image for this issue. The actual cover is not yet available at this time.) This is an open access article which appeared in a journal publishedby Elsevier. This article is free for everyone to access, download and read.Any restrictions on use, including any restrictions on furtherreproduction and distribution, selling or licensing copies, or postingto personal, institutional or third party websites are defined by theuser license specified on the article.For more information regarding Elsevier's open access licensesplease visit: http://www.elsevier.com/openaccesslicenses  Mini Review A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control Chunhong Long a , E. Chao a , Lin-Tai Da b , Jin Yu a, ⁎ a Beijing Computational Science Research Center, Beijing, 100193, China b Shanghai Center for Systems Biomedicine, Shanghai JiaoTong University, Shanghai 200240, China a b s t r a c ta r t i c l e i n f o  Article history: Received 1 March 2019Received in revised form 25 April 2019Accepted 4 May 2019Available online 09 May 2019 RNApolymerase(RNAP)frombacteriophageT7isarepresentativesingle-subunitviralRNAPthatcantranscribewith high promoter activities without assistances from transcription factors. We accordingly studied this smalltranscription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and  fi delity control in the RNAP transcription elongation. Here we summarize our computa-tional work from several recent publications to demonstrate  fi rst how T7 RNAP translocates via Brownian alikemotionsalongDNArightafterthecatalyticproductrelease.Thenweshowhowthebackwardtranslocationmo-tionsarepreventedatpost-translocationuponsuccessfulnucleotideincorporation,whichisalsosubjecttostep-wise nucleotide selection and acts as a pawl for  “ selective ratcheting ” . The structural dynamics and energeticsfeatures revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet workingscenario of a prototypical transcription machine with sophisticated nucleotide selectivity for  fi delity control.The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPsand some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machineryin higher organisms.© 2019 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and StructuralBiotechnology.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: RNA polymerasePPi releaseTranslocationNucleotide selectionFidelity control Contents 1. T7 RNA Polymerase as a Minimal Transcription Machine Model System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6382. PPi Product Release Unlikely Drives the Translocation of T7 RNAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6393. Translocation Proceeds in Brownian Motions and is Facilitated bythe O-helix Fluctuation Opening at Pre-translocation that may also PreventBacktracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6394. Selective Ratcheting Starts From the Nucleotide Pre-insertion Checkpoint. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6415. Selective Ratcheting Proceeds Through Slow Nucleotide Insertion With Substantial Selection . . . . . . . . . . . . . . . . . . . . . . . . . . 6426. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643 1. T7 RNA Polymerase as a Minimal Transcription Machine ModelSystem The RNA polymerase (RNAP) from bacteriophage T7 is regarded asone of the smallest transcription machines [1 – 3]. In bacteria and eu-karyotespecies,RNAPII,thecoreengineofthetranscriptionmachinery,workswithavarietyoftranscriptionfactorstosupportgeneexpression[4 – 8].RNAPIIitselfconsistsofmultiplepolypeptides,i.e.,maintainingacomplex molecular architecture. In comparison, T7 RNAP is asingle-subunit enzyme with a simple hand-like structure [9 – 12], andit is capable of transcribing with high promoter activity or processivity,self-suf  fi ciently, without assistances from transcription factors, frominitiationtoelongationandtotermination.Indeed,T7RNAPstructurallyresembles a wide class of DNA polymerases (DNAPs), along with someother viral and mitochondrial RNAP species [10,13]. Hence, T7 RNAP makes a minimal model system to study transcription. Computational and Structural Biotechnology Journal 17 (2019) 638 – 644 ⁎  Corresponding author. E-mail address:  jinyu@csrc.ac.cn (J. Yu).https://doi.org/10.1016/j.csbj.2019.05.0012001-0370/© 2019 The Authors. Publishedby Elsevier B.V.on behalfof Research NetworkofComputational andStructural Biotechnology. This isan openaccess articleunder theCCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/csbj  The high-resolution crystal structures of T7 RNAP had been initiallydetermined by Sousa et al. [14] and then by Thomas A Steitz lab co-workers on its transcription initiation to elongation complexes sincethe late last century [15 – 18]. In particular, several states of T7 RNAPelongation complex have been obtained, from nucleotide insertion orsubstrate state to catalytic product state, and to post-translocationstate, together with an additional pre-insertion complex then resolvedby Temiakov et al. [19]. Meanwhile, extensive bio-chemical studies[20 – 23]alongwithsingle-moleculemeasurementsonT7RNAPelonga-tion[24 – 27]providesubstantialquantitativefeaturesoftheenzymeki-netics,frominitiationtoelongation.Accordingly,physicalmodelingandmoleculardynamics(MD)simulationonthissmallesttranscriptionma-chine became feasible, so that to reveal underlying molecular mecha-nisms and essential structural dynamics details.We have recently studied transcription elongation of T7 RNAP bycombining physical modeling and all-atom MD simulations, addressingboth mechano-chemical couplingand fi delity control mechanisms dur-ingelongation[28 – 36].Themechanochemistryconcernsabouthowtheproteinmachineutilizeschemicalfreeenergytogeneratemechanicalordirectional motions, referring to how the chemical synthesis of RNAcouples with the polymerase translocation along DNA in the RNAP sys-tem. T7 RNAP had been suggested to work via a  ‘ power stroke ’  (PS)mechanism [17,37,38], in which product release directly drives the RNAP translocation via simultaneous protein subdomain opening.Meanwhile, T7 RNAP along with RNAPs from bacteria and eukaryoticspecies had also been proposed to function in a  ‘ Brownian ratchet ’ (BR) scenario [21,25,39 – 42]. Below, we elaborate on how our studiesactually support the BR working scenario of T7 RNAP, in which thetranslocation proceeds in Brownian motions after the product release,whiletheratchetingpartisful fi lledlargelybycognatenucleotideincor-porationtothegrowingendofthesynthesizingRNA.Sincenon-cognatenucleotides unlikely support successful nucleotide incorporation orratcheting, one regards that nucleotide selection plays a crucial rolefor the BR scenario such that an RNAP actually conducts  ‘ selectiveratcheting ’  along DNA. Accordingly, we illustrate then how T7 RANPachieves the nucleotide selection for the transcription  fi delity control.Indeed,themechanismscanberepresentativeorapplyingeneraltore-lated enzymes on the catalyzed polymerization processes, in the pres-ence of molecular template, though speci fi c structural elements dovary for different polymerization machines. 2.PPi ProductRelease Unlikely Drivesthe Translocation of T7 RNAP In previous structural studies of T7 RNAP, suggestions had beenmade on thePS mechanism suchthat thepyrophosphate (PPi) productrelease after catalysis directly drives the translocation via rotationalopening of the  fi ngers subdomain [17]. On the other hand, early [39] and immediately later single-molecule force measurements on T7RNAP suggested alternatively the BR scenario [25]. Accordingly, we in-vestigated the mechano-chemical coupling by studying the PPi release fi rst, using atomistic MD simulation [31].Indeed,the PPi release step alongwith thetranslocation of RNAP onDNA turns out to be too fast to be monitored directly by experiments.For example, the single molecule measurements had shown that forcesimplemented to hinder the RNAP movements on the DNA hardly slowdown the overall elongation, suggesting that the translocation is not arate-limiting step during an elongation cycle [25,26]. The elongation cycle of T7 RNAP, however, lasts tens of milliseconds or longer [22].One can accordingly estimate that the fast steps of the product releaseand translocation happen from microseconds to sub-milliseconds[43 – 45], which are nevertheless too long for straightforward computa-tional samplings by the atomistic MD simulations.The all-atom simulation systems of T7 RNAP-DNA-RNA complexwith explicit water solvent include over 100 K atoms. For systems of such a size, one can routinely simulate up to several microsecondsunder current high-performance computing technologies; yet it is stillcomputationally prohibiting to further approach over tens of microsec-ondstomillisecondtimescale.Fortunately,bylaunchingextensivesub-microsecondsequilibriumsimulationsthatspreadaroundawiderangeof conformation space for the relevant process, we were able to con-struct the Markov-state model (MSM) for the PPi release, and later forthe translocation of T7 RNAP, which are estimated to happen at tensof microsecond time scale [31,34]. The strength and technical issues in building the MSM using MD can be found in abundant literature else-where [46 – 50].The MSM we constructed (two hundreds micro-states according tostructural root-mean-square deviations or RMSDs, and three macro-states for visualization) shows a jump-from-cavity PPi release mecha-nism (see Fig. 1A), in which the PPi-bound product state (S1a) and apre-activationintermediatestate(S1b)dominatetheoverallpopulation(90%),whilethePPireleasedstate(S2)isachievedbythermallyactivat-ing transitions S1a → S1b → S2 [31]. Inside the  ‘ cavity ’  around the activesite, PPi is hold by two aspartate residues (Asp527 and Asp812) thatare crucial for the catalysis. Then PPi can shift away and associatemore closely with positively charged residues aligning the product re-lease channel (e.g. Lys631 and Arg627), particularly with Lys472 thatis key to assist PPi to  ‘  jump ’  out of the cavity via the lysine side-chainswing or fl uctuations. Interestingly,there isalways a lysineor a homol-ogousargininelocatedattheexitoftheproductreleasechannelinotherpolymerase species (including yeast RNAP II, bacterial RNAP, humanmitochondrial RNAP, and several species of DNAPs), which appears toassist the PPi release in general [31]. Note that the jump-from-cavityhappens comparatively slowly at the S1b → S2 transition, which is esti-mated above several microseconds at least.Essentially,the fi ngerssubdomaindoesnotshowsubstantialconfor-mational changes during the many short equilibrium simulation pro-cesses for the PPi release. Even in comparatively long simulations, the fi ngers subdomain or the O-helix on thesubdomain shows nosubstan-tial opening, either in the PPi initially bound state, or upon PPi removalor its charge neutralization for control, up to a microsecond time scale.Anyhow, the rotational  fl uctuations of the O-helix increase in the con-trol simulations, once having PPi or its charge removed. Hence, itseems that the PPi release does not necessarily couple with a progres-sive rotational opening of the O-helix or the  fi ngers subdomain, whichisessentialtocompletetheRNAPtranslocation.Besides,thethermalac-tivation of the PPi release also suggests that energetically it is unlikelyfor the release process to directly power or drive the translocation. In-stead, the PPi release likely only enhances the rotational  fl exibilities of the  fi ngers subdomain, which then facilitates the RNAP translocationthereafter. 3. Translocation Proceeds in Brownian Motions and is Facili-tated bythe O-helix Fluctuation Opening at Pre-translocationthat may also Prevent Backtracking  ThenweemployedevenmoreextensiveMDsimulationsinaggrega-tion to ~ 10  μ  s to construct the MSM of the T7 RNAP translocation onDNA, by clustering a large amount of simulation snapshots into 500micro-states, according to time-structure independent componentanalysis(tICA)[51,52].Theresultedmodelisfurthersimpli fi edforvisu-alization into a six-state macro-state representation (see Fig. 1C) [34]. In the six-state translocation network model representation, boththe O-helix and Y-helix on the  fi ngers subdomain play signi fi cantroles, and they show rotational opening motions in non-synchronizedmanner. Importantly, in the initial pre-translocation state (S1), boththe O-helix and Y-helix show a  ‘ semi-open ’  conformation on average(i.e.,therotationanglepeaked~15°),withsigni fi cantwide fl uctuationsspanning from the closed conformations to open ones ([34]. Microsec-ond transition into a less-populated pre-translocation con fi guration(S2) allows base un-stacking of the transition nucleotide (TN) fromF644 (on the Y-helix) yet somehow quenches the O/Y-helix openingto the close status. The Y-helix opens  fi rst in the transition state (S3), 639 C. Long et al. / Computational and Structural Biotechnology Journal 17 (2019) 638 – 644  after that the O-helix opens (S4 to S6). Essentially, Y639 (on the O-helix) pushes onto the 3 ′ -end of the RNA to allow it to move ahead of the template DNA (S3). Hence, the O-helix opening seems to well cou-ple with theDNA forward translocation.Overall, thefree energy pro fi leof translocation appears comparatively  fl at so that Brownian motionsdominate. The slowest step of the translocation takes place in the tran-sitiontothekeyintermediatestate(S3),whichisestimatedtolast overtens of microseconds at least.Interestingly, the O-helix along with the Y-helix (or say the  fi ngerssubdomain) seems to be able to open by enhanced rotational oscilla-tions or  fl uctuations (after the PPi release) in the pre-translocationstate. Note that it is NOT progressive opening yet until toward thepost-translocation state. The product crystal structure with PPi boundwas captured in the O-helix (or  fi ngers subdomain) closed con fi gura-tion [17], so that one expects that: (i) If the O-helix opens right afterthe PPi release, it is an indication that the translocation can be drivenbytheO-helixopening(asinthePSscenario),orelse(ii)TheO-helixre-mains closed at pre-translocation after the PPi release, and then opensonly after the translocation (consistent with the BR scenario, but notnecessarily the only situation). However, both the statements aremore or less inconsistent with our observations. Our studies indicatethattherotational fl exibilityoftheO-helixorthe fi ngerssubdomainbe-comes high immediately after the PPi release [30], so that one shouldtreat the rotation angle as a highly  fl uctuating random variable(i.e., with a non-trivial probability distribution), rather than a  fi xedvalue. The occasional oscillations to open of the O-helix atpre-translocation appear to be crucial to facilitate the RNAP forwardtranslocation,i.e.,byloweringtheactivationbarrierofthetranslocation.Meanwhile, we suspected that the occasional O-helix openings in thepre-translocation state might even prevent backtracking in T7 RNAP[34].ItisthentheaveragerotationaldegreeoftheO-helixorthe fi ngerssubdomainthatpersistentlyshiftsfromtheclosedtoopenfromthepre-to post-translocation state. Previous studies had also concerned aboutrotational fl exibilitiesofthethumbsubdomain[53,54].Wealsonoticed substantial rotational movements (~25°; non-published results) of thethumb subdomain from the pre- to the post-translocation.Backtracking turns out to be an ef  fi cient way of coordination forRNAP todealwitherrors of nucleotide incorporation,i.e., by proofread-ing or editing; or it supports necessary pauses during the transcriptionelongation,e.g., to coordinatewith translationbyribosome[55 – 57].Al-though backtracking had been identi fi ed in multi-subunit RNAPs oreven the single subunit mitochondrial RNAP (mtRNAP) [58,59], it has not been detected for T7 RNAP. We then hypothesized that the verymechanism to facilitate the translocation, i.e., the O-helix  fl uctuationto opening in the pre-translocation state, may also prevent T7 RNAPbacktracking. To test the hypothesis, we computationally designed a Fig.1. ThePPireleaseandtranslocationmechanismofT7RNAPrevealedfromextensiveMDsimulationsandtheMSMconstruction[31,34].(A)Leftpanel:AmolecularimageofT7RNAP elongation product complex with PPi bound (PDB:1S77) [17]; Rightpanel: The three-state MSMof the PPi release process derived from 100 × 20 ns MD simulations(by clustering ~10 6 conformationsinto200microstatesetc.)[31].NotethatthePPigroupisdepictedinredspheres,whiletheO-helixiscoloredgreen.(B)TheschematicsofanincompleteBrownian-ratchetdevice, which is still lack of the  ‘ pawl ’ . (C) The six-state MSM of the RNAP translocation on the DNA (129 × 80 ns all-atom MD simulation, clustering ~ 9 × 10 5 conformations into 500microstates etc.) [34]. The translocation starts after the PPi release from the product complex, or the pre-translocation state (S1), transiting all the way (via S2-S5 and mainly S3) tothe post-translocation state (S6; PDB: 1MSW) [16] (populations and transition rates are labeled). Note that both the O-helix (green) and Y-helix (cyan) on the  fi ngers subdomain areshown (with open/closed labeled), along with Y639 and F644 that are key residues in the translocation. The RNA and template DNA nucleotides are colored in blue and red,respectively. (D) The probability distributions of the rotational angles of the O-helix and the Y-helix during translocation process (from S1 to S6) are presented (as taken from [34]).(For interpretation of the references to colour in this fi gure legend, the reader is referred to the web version of this article.)640  C. Long et al. / Computational and Structural Biotechnology Journal 17 (2019) 638 – 644  mutantT7RNAPwithseveralresiduesreplacedontheO-helixtomimicthemtRNAP that is structurally similar to T7 RNAP [60].Our simulationresultsshowedthatthemutantT7RNAPwouldhavetheO-helixclosedupon the 3 ′ -end of the RNA being pulled to initiate the backtrackingfromthepre-translocation,whiletheO-helixopensfor sucharesponseinthewild-typeT7RNAP[34].Hence,weconsideredthatthemutantT7RNAP we made might be able to backtrack to some extent. PreliminaryexperimentalstudiessupportedthehypothesissuchthatthemutantT7RNAP maintained transcription activities, but lower than the wild-typesystem [34]. Further experimental investigation is still needed, e.g., at asingle molecule level, to con fi rm on the mutant and wild-type T7RNAPcapabilities of backtracking. 4. Selective Ratcheting Starts From the Nucleotide Pre-insertionCheckpoint In an RNAP elongation cycle, the incoming nucleoside triphosphate(NTP) is recruited into the RNAP enzyme active site according to theWatson-Crick (WC) base pairing with the template DNA nucleotide.The RNAP translocation allows the incorporated nucleotide at the 3 ′ -end of the synthesizing RNA to move upstream to vacant the activesite. According to the BR scenario, prior to the NTP association, RNAPcan keep moving forward and backward on DNA, due to the nearly fl at free energy surface of the translocation. Once the incoming NTPbinds and occupies the active site, the backward movement of RNAPcan be prevented, so that forward translocation is  fi nally biased uponthe full nucleotide incorporation. That says, the NTP association andincorporationactasa ‘ pawl ’ inaratchetdevicetoachievetheBRprocess(seeFigs.2Aand1B)[61].Nevertheless,non-cognatenucleotidespecies mayalsobindbutlikelydissociateprematurelybeforechemicalsynthe-sis,duetothenucleotideselectivityconductedbyRNAPforthepurposeof   fi delity control. Consequently, only those successfully incorporatednucleotides, or in principle, the cognate nucleotide species, contributeto the ratcheting of RNAP along DNA.A prominent feature of the nucleotide addition cycle (NAC) of T7RNAPandrelatedsingle-subunitpolymerasespeciesisthatanucleotidepre-insertionstateexists[12,19,62,63],andourstudiescon fi rmthatthepre-insertion state serves well as an initial kinetic checkpoint to screennon-cognate nucleotidespeciesout of theactive site[30,64,36].Indeed, the pre-insertion complex of T7 RNAP in a  ‘ semi-open ’  conformationhad been crystalized with a cognate nucleotide bound to a pre-insertion site, slightly away from the active site [19]. Although the WCbase pairinghad not been well captured between the pre-insertion nu-cleotide and the template counterpart in the crystal structure, our MDsimulation on the pre-insertion complex revealed the WC base pairingformation after ~ 50 ns equilibrium simulation [30]. Besides, when wereplaced the cognate nucleotide (rATP) by the non-cognate species(rGTP, dATP etc.) from the pre-insertion crystal structure complex andconducted equilibrium simulations accordingly, we found that thenon-cognate nucleotide would be  ‘ grabbed ’  by Y639, which actuallyblocks the insertion site of the DNA template nucleotide (i.e., thetransition nucleotide or TN) in the pre-insertion complex. In such anequilibrated rGTP pre-insertion complex (see the rGTP  off-path  pre-insertionstructureinFig.2B upperright  ,orFig.2Dcon fi g1inthemiddle Fig.2. SelectiveratchetingofT7RNAPonDNAasnucleotidesaredifferentiatedandselectedasbeingincorporated tothegrowingendofRNAinsynthesis.(A)Aschematicsshowingfreeenergy pro fi les of the cognate/right and non-cognate/wrong nucleotide addition cycle (NAC), which includes translocation, nucleotide binding/pre-insertion, nucleotide insertion,catalysis, and PPi product release. The nucleotide pre-insertion, insertion, and catalysis together serve as a  ‘ pawl ’  for the ratchet. (B) The molecular views around the active site of thepre-insertion complexes modeled for our simulation studies [30,64]. Upper row: the non-cognate rGTP pre-insertion complexes, made  on-path  and  off-path  [64], respectively; Lowerrow: the cognate rATP and the non-cognate dATP ( off-path ) pre-insertion complexes [30]. (C) The free energy pro fi les or PMFs calculated from umbrella sampling simulations of thecognate rATP and non-cognate rGTP  off-path  insertion [36]. (D) The molecular views around the active site of T7 RNAP from representative snapshots captured in the umbrellasampling MD simulations of cognate rATP, non-cognate rGTP ( off-path ) and dATP ( on-path ) insertion process [36]. See text for further illustration.641 C. Long et al. / Computational and Structural Biotechnology Journal 17 (2019) 638 – 644
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