- Email: [email protected]
The Ins and Outs of Viral RNA Polymerase Translocation
Commentary
The Ins and Outs of Viral RNA Polymerase Translocation
David D. Boehr Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA
Correspondence to David D. Boehr:107 Chemistry Building, The Pennsylvania State University, University Park, PA 16802, USA. DOI of original article: http://dx.doi.org/10.1016/j.jmb.2013.12.031 http://dx.doi.org/10.1016/j.jmb.2013.12.030 Edited by A. Pyle
Central to the replication of RNA viruses is the RNA-dependent RNA polymerase (RdRp) [1], an important antiviral drug target [2–4]. RdRps belong to a large superfamily of template-directed nucleic acid polymerases, including DNA polymerases and reverse transcriptases [1]. These polymerases have a similar structural fold, which has been described as a “cupped right hand” with fingers, thumb and palm subdomains [5] (Fig. 1), and a similar chemical mechanism, involving at least two metal ions [6]. Polymerase function can be divided into three phases, including nucleotide selection, phosphodiester bond formation and translocation to the next templating nucleobase to prepare for the next round of nucleotide addition. Structural biology has provided tremendous insight into the structural changes that accompany each stage of catalysis for multiple classes of polymerases [7–10]. Least understood is how conformational changes in the polymerases may couple to translocation. In A-family DNA polymerases, a partial opening of the active site involving the temporary displacement of the “O-helix” and other regions in the fingers subdomain is likely important for movement along the DNA [11– 15]. Such a mechanism is unlikely to aid translocation in RdRps; these polymerases contain an extension of the fingers through the “fingertips” that interacts with the thumb domain, likely precluding a similar “opening” of the active site [16] (Fig. 1). In this issue, Sholders and Peersen have provided tantalizing insight into an alternative mechanism for translocation in RdRps, involving a palm structural motif known as “motif-B” [17]. Steric clashes between the motif-B loop and the template RNA may promote movement along the RNA to the next catalytic register. These findings are especially intriguing considering that the motif-B loop is involved in multiple stages of RdRp catalysis and
may be a master regulator of polymerase function [18]. Recent years have brought tremendous structural insight into RdRps [9,10,19]. The Peersen laboratory, in particular, has been able to solve multiple X-ray crystal structures of poliovirus (PV) and other viral RdRps in elongation complexes [20–22], enabling them to capture snapshots of the RdRp before and after nucleotide addition. In contrast to some observations in DNA polymerases, the conformational changes in RdRps observed by X-ray crystallography are rather subtle. There are no grand conformational changes in the fingers and thumb subdomains, but instead, activation of the enzyme requires localized changes to the palm subdomain to reposition key residues important for nucleotide binding and/or phosphodiester bond formation [22]. Correct nucleotide binding induces a re-alignment of β-strands in the palm subdomain, including structural motif-A and motif-C, resulting in the repositioning of the absolutely conserved Asp233 (PV numbering) to allow interactions with both metal ions required for RdRp function [22]. This conformational change is likely triggered when motif-B residues Ser288 and Asn297 make hydrogen-bonding interactions with the ribose hydroxyls of the incoming nucleotide. In this way, conformational changes in the RdRp efficiently couple nucleotide selection to phosphodiester bond formation. In their current manuscript, Sholders and Peersen capture residues 288–292 in the motif-B loop in three major conformations, which they term “in/up”, “in/ down” and “out/down”. The “in/out” designation is based on whether Cys290 is buried “in” a hydrophobic pocket directly behind the loop or is “out” of the pocket and exposed to solvent. The “up/down” designation refers to the orientation of the Ser288 side chain, whether it points “up” toward the ring
0022-2836/$ - see front matter © 2014 The Author. Published by Elsevier Ltd. All rights reserved.
J. Mol. Biol. (2014) 426, 1373–1376
1374
Viral RNA Polymerase Translocation
Fig. 1. Structural dynamics of motif-B drive RdRp function. (a) Structure of the Norwalk virus RdRp (PDB ID 1SH3) showing the canonical right-hand cupped structures of RdRp with fingers, thumb and palm subdomains and highlighting palm structural motif-A (red), motif-B (blue), motif-C (magenta) and motif-D (orange). The motif-B loop is circled. (b) RdRps show structural variability in the motif-B loop, likely important for function, including RNA translocation.
finger or “down” toward the active site. In their model, nucleotide binding induces the “in/up” to “in/down” transition, which repositions Ser288 and begins the cascade of structural rearrangements, including changes in the palm motifs, required to catalyze phosphodiester bond formation. Following the chemical step, they propose the motif-B loop to transition from “in/down” to “out/down”. The “out/down” conformation would result in a steric clash between the motif-B loop and the backbone of the RNA template strand. Relief of this steric clash might help to facilitate translocation along the RNA and/or prevent backtracking once translocated. Functional analyses of motif-B site mutants provide further evidence of the importance of these conformations in RdRp catalysis. Perhaps most intriguing is the finding that the G289A variant can catalyze single nucleotide addition, but processive elongation activity is completely abolished. One explanation is that the G289A substitution prevents translocation necessary for processive activity by locking the enzyme in an “in/up” conformation. What triggers the change to the “out/down” motif-B conformation remains poorly understood. Studies from other nucleic acid polymerases, including DNA polymerases [8] and multi-subunit RNA polymerases [23,24], have led to two major models of how translocation is coordinated with other stages of polymerase function. In the “active” or “powerstroke” model, nucleotide hydrolysis and the release of pyrophosphate (PPi) provide the energy for translocation [13,25,26]. In contrast, the “passive” or “Brownian ratchet” model proposes that, following nucleotide addition, the enzyme can thermally fluctuate between a pre-translocated and a posttranslocated state and binding of the next incoming nucleotide traps the post-translocated state, helping to drive the polymerase along the DNA/RNA [27– 30]. These models are not necessarily mutually
exclusive; conformational changes may be triggered by the release of pyrophosphate, but translocation may not be completed until the binding of the next nucleotide. The protonation state of the polymerases may also help to coordinate phosphodiester bond formation with translocation. In yeast RNA polymerase II, a multi-subunit polymerase structurally distinct from RdRps, it has been proposed that His1085 on the “trigger loop” acts as a general acid to protonate the β-phosphate of the incoming nucleotide to generate a better pyrophosphate-leaving group [31]. The release of pyrophosphate and/or change in the protonation state of His1085 may lead to conformational changes that drive translocation. Indeed, conformational changes in the trigger loop can induce further structural changes to the “bridgehelix”, which is important for translocation [31]. In the A-family DNA polymerases, the general acid is an absolutely conserved Lys on the O-helix [32]. Again, changes in the protonation state of the general acid may couple to structural changes in the O-helix important for translocation. The general acid in RdRps has been proposed to be an absolutely conserved Lys in the motif-D loop [32]. It was previously demonstrated that the conformational state of PV RdRp depends on the protonation state of the motif-D Lys [33], and a long-range interaction network that would be able to communicate structural dynamic changes from motif-D to motif-B has been proposed [34]. Molecular dynamic simulations have also suggested that motions in the motif-D loop are coupled to fluctuations in motif-A and other regions in the active site [35], which might be important for governing the motif-B conformational state. The function of the general acid is closely tied to translocation, considering that amino acid substitutions at the general acid substantially reduce processivity [32].
Viral RNA Polymerase Translocation
Other biophysical studies will likely be required to delineate the sequence of events in RdRp translocation. Single-molecule studies have proven especially revealing in other polymerases and generally support the “passive” model of translocation [36–38]. NMR studies may also reveal the capacity for the RdRp to fluctuate between pre-translocated and post-translocated states [39]. Further X-ray crystallography studies in the presence of excess pyrophosphate and 3′ deoxy RNA may reveal other structural states important in the translocation process. The site mutants studied by Sholders and Peersen should serve as important tools and starting points in the further unraveling of the translocation mechanism and its relationship to RNA replication speed and fidelity.
Acknowledgements I thank Drs. Jamie Arnold and Craig Cameron for their insightful comments.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercialShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
References [1] Ng KK, Arnold JJ, Cameron CE. Structure–function relationships among RNA-dependent RNA polymerases. Curr Top Microbiol Immunol 2008;320:137–56. [2] Waheed Y, Bhatti A, Ashraf M. RNA dependent RNA polymerase of HCV: a potential target for the development of antiviral drugs. Infect Genet Evol 2013;14:247–57. [3] Malet H, Masse N, Selisko B, Romette JL, Alvarez K, Guillemot JC, et al. The flavivirus polymerase as a target for drug discovery. Antiviral Res 2008;80:23–35. [4] Vignuzzi M, Stone JK, Andino R. Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications. Virus Res 2005;107: 173–81. [5] Ollis DL, Brick P, Hamlin R, Xuong NG, Steitz TA. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 1985;313:762–6. [6] Steitz TA. A mechanism for all polymerases. Nature 1998;391:231–2. [7] Berdis AJ. Mechanisms of DNA polymerases. Chem Rev 2009;109:2862–79. [8] Steitz TA. Visualizing polynucleotide polymerase machines at work. EMBO J 2006;25:3458–68. [9] Ferrer-Orta C, Agudo R, Domingo E, Verdaguer N. Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr Opin Struct Biol 2009;19:752–8.
1375 [10] Lescar J, Canard B. RNA-dependent RNA polymerases from flaviviruses and Picornaviridae. Curr Opin Struct Biol 2009;19:759–67. [11] Cheetham GM, Jeruzalmi D, Steitz TA. Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature 1999;399:80–3. [12] Yin YW, Steitz TA. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 2002;298:1387–95. [13] Yin YW, Steitz TA. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 2004;116:393–404. [14] Li Y, Korolev S, Waksman G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J 1998;17:7514–25. [15] Golosov AA, Warren JJ, Beese LS, Karplus M. The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis. Structure 2010;18:83–93. [16] Hansen JL, Long AM, Schultz SC. Structure of the RNAdependent RNA polymerase of poliovirus. Structure 1997;5:1109–22. [17] O'Reilly EK, Kao CC. Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology 1998;252:287–303. [18] Garriga D, Ferrer-Orta C, Querol-Audi J, Oliva B, Verdaguer N. Role of motif B loop in allosteric regulation of RNA-dependent RNA polymerization activity. J Mol Biol 2013;425:2279–87. [19] McDonald SM, Tao YJ, Patton JT. The ins and outs of fourtunneled Reoviridae RNA-dependent RNA polymerases. Curr Opin Struct Biol 2009;19:775–82. [20] Gong P, Kortus MG, Nix JC, Davis RE, Peersen OB. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts. PLoS One 2013;8:e60272. [21] Kortus MG, Kempf BJ, Haworth KG, Barton DJ, Peersen OB. A template RNA entry channel in the fingers domain of the poliovirus polymerase. J Mol Biol 2012;417:263–78. [22] Gong P, Peersen OB. Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A 2010;107:22505–10. [23] Svetlov V, Nudler E. Macromolecular micromovements: how RNA polymerase translocates. Curr Opin Struct Biol 2009;19:701–7. [24] Kireeva M, Kashlev M, Burton ZF. Translocation by multisubunit RNA polymerases. Biochim Biophys Acta 2010;1799:389–401. [25] Temiakov D, Patlan V, Anikin M, McAllister WT, Yokoyama S, Vassylyev DG. Structural basis for substrate selection by t7 RNA polymerase. Cell 2004;116:381–91. [26] Wang HY, Elston T, Mogilner A, Oster G. Force generation in RNA polymerase. Biophys J 1998;74:1186–202. [27] Landick R. Active-site dynamics in RNA polymerases. Cell 2004;116:351–3. [28] Guajardo R, Sousa R. A model for the mechanism of polymerase translocation. J Mol Biol 1997;265:8–19. [29] Bar-Nahum G, Epshtein V, Ruckenstein AE, Rafikov R, Mustaev A, Nudler E. A ratchet mechanism of transcription elongation and its control. Cell 2005;120:183–93. [30] von Hippel PH. An integrated model of the transcription complex in elongation, termination, and editing. Science 1998;281:660–5.
1376 [31] Wang D, Bushnell DA, Westover KD, Kaplan CD, Kornberg RD. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 2006;127:941–54. [32] Castro C, Smidansky ED, Arnold JJ, Maksimchuk KR, Moustafa I, Uchida A, et al. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat Struct Mol Biol 2009;16:212–8. [33] Yang X, Smidansky ED, Maksimchuk KR, Lum D, Welch JL, Arnold JJ, et al. Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition. Structure 2012;29:1519–27. [34] Yang X, Welch JL, Arnold JJ, Boehr DD. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance. Biochemistry 2010;49:9361–71. [35] Moustafa IM, Shen H, Morton B, Colina CM, Cameron CE. Molecular dynamics simulations of viral RNA-dependent
Viral RNA Polymerase Translocation
[36]
[37]
[38]
[39]
RNA polymerases link conserved and correlated motions of functional elements to fidelity. J Mol Biol 2011;410:159–81. Dahl JM, Mai AH, Cherf GM, Jetha NN, Garalde DR, Marziali A, et al. Direct observation of translocation in individual DNA polymerase complexes. J Biol Chem 2012;287:13407–21. Lieberman KR, Dahl JM, Mai AH, Akeson M, Wang H. Dynamics of the translocation step measured in individual DNA polymerase complexes. J Am Chem Soc 2012;134:18816–23. Dangkulwanich M, Ishibashi T, Liu S, Kireeva ML, Lubkowska L, Kashlev M, et al. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. Elife 2013;2:e00971. Loria JP, Berlow RB, Watt ED. Characterization of enzyme motions by solution NMR relaxation dispersion. Acc Chem Res 2008;41:214–21.
The Ins and Outs of Viral RNA Polymerase Translocation
David D. Boehr Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA
Correspondence to David D. Boehr:107 Chemistry Building, The Pennsylvania State University, University Park, PA 16802, USA. DOI of original article: http://dx.doi.org/10.1016/j.jmb.2013.12.031 http://dx.doi.org/10.1016/j.jmb.2013.12.030 Edited by A. Pyle
Central to the replication of RNA viruses is the RNA-dependent RNA polymerase (RdRp) [1], an important antiviral drug target [2–4]. RdRps belong to a large superfamily of template-directed nucleic acid polymerases, including DNA polymerases and reverse transcriptases [1]. These polymerases have a similar structural fold, which has been described as a “cupped right hand” with fingers, thumb and palm subdomains [5] (Fig. 1), and a similar chemical mechanism, involving at least two metal ions [6]. Polymerase function can be divided into three phases, including nucleotide selection, phosphodiester bond formation and translocation to the next templating nucleobase to prepare for the next round of nucleotide addition. Structural biology has provided tremendous insight into the structural changes that accompany each stage of catalysis for multiple classes of polymerases [7–10]. Least understood is how conformational changes in the polymerases may couple to translocation. In A-family DNA polymerases, a partial opening of the active site involving the temporary displacement of the “O-helix” and other regions in the fingers subdomain is likely important for movement along the DNA [11– 15]. Such a mechanism is unlikely to aid translocation in RdRps; these polymerases contain an extension of the fingers through the “fingertips” that interacts with the thumb domain, likely precluding a similar “opening” of the active site [16] (Fig. 1). In this issue, Sholders and Peersen have provided tantalizing insight into an alternative mechanism for translocation in RdRps, involving a palm structural motif known as “motif-B” [17]. Steric clashes between the motif-B loop and the template RNA may promote movement along the RNA to the next catalytic register. These findings are especially intriguing considering that the motif-B loop is involved in multiple stages of RdRp catalysis and
may be a master regulator of polymerase function [18]. Recent years have brought tremendous structural insight into RdRps [9,10,19]. The Peersen laboratory, in particular, has been able to solve multiple X-ray crystal structures of poliovirus (PV) and other viral RdRps in elongation complexes [20–22], enabling them to capture snapshots of the RdRp before and after nucleotide addition. In contrast to some observations in DNA polymerases, the conformational changes in RdRps observed by X-ray crystallography are rather subtle. There are no grand conformational changes in the fingers and thumb subdomains, but instead, activation of the enzyme requires localized changes to the palm subdomain to reposition key residues important for nucleotide binding and/or phosphodiester bond formation [22]. Correct nucleotide binding induces a re-alignment of β-strands in the palm subdomain, including structural motif-A and motif-C, resulting in the repositioning of the absolutely conserved Asp233 (PV numbering) to allow interactions with both metal ions required for RdRp function [22]. This conformational change is likely triggered when motif-B residues Ser288 and Asn297 make hydrogen-bonding interactions with the ribose hydroxyls of the incoming nucleotide. In this way, conformational changes in the RdRp efficiently couple nucleotide selection to phosphodiester bond formation. In their current manuscript, Sholders and Peersen capture residues 288–292 in the motif-B loop in three major conformations, which they term “in/up”, “in/ down” and “out/down”. The “in/out” designation is based on whether Cys290 is buried “in” a hydrophobic pocket directly behind the loop or is “out” of the pocket and exposed to solvent. The “up/down” designation refers to the orientation of the Ser288 side chain, whether it points “up” toward the ring
0022-2836/$ - see front matter © 2014 The Author. Published by Elsevier Ltd. All rights reserved.
J. Mol. Biol. (2014) 426, 1373–1376
1374
Viral RNA Polymerase Translocation
Fig. 1. Structural dynamics of motif-B drive RdRp function. (a) Structure of the Norwalk virus RdRp (PDB ID 1SH3) showing the canonical right-hand cupped structures of RdRp with fingers, thumb and palm subdomains and highlighting palm structural motif-A (red), motif-B (blue), motif-C (magenta) and motif-D (orange). The motif-B loop is circled. (b) RdRps show structural variability in the motif-B loop, likely important for function, including RNA translocation.
finger or “down” toward the active site. In their model, nucleotide binding induces the “in/up” to “in/down” transition, which repositions Ser288 and begins the cascade of structural rearrangements, including changes in the palm motifs, required to catalyze phosphodiester bond formation. Following the chemical step, they propose the motif-B loop to transition from “in/down” to “out/down”. The “out/down” conformation would result in a steric clash between the motif-B loop and the backbone of the RNA template strand. Relief of this steric clash might help to facilitate translocation along the RNA and/or prevent backtracking once translocated. Functional analyses of motif-B site mutants provide further evidence of the importance of these conformations in RdRp catalysis. Perhaps most intriguing is the finding that the G289A variant can catalyze single nucleotide addition, but processive elongation activity is completely abolished. One explanation is that the G289A substitution prevents translocation necessary for processive activity by locking the enzyme in an “in/up” conformation. What triggers the change to the “out/down” motif-B conformation remains poorly understood. Studies from other nucleic acid polymerases, including DNA polymerases [8] and multi-subunit RNA polymerases [23,24], have led to two major models of how translocation is coordinated with other stages of polymerase function. In the “active” or “powerstroke” model, nucleotide hydrolysis and the release of pyrophosphate (PPi) provide the energy for translocation [13,25,26]. In contrast, the “passive” or “Brownian ratchet” model proposes that, following nucleotide addition, the enzyme can thermally fluctuate between a pre-translocated and a posttranslocated state and binding of the next incoming nucleotide traps the post-translocated state, helping to drive the polymerase along the DNA/RNA [27– 30]. These models are not necessarily mutually
exclusive; conformational changes may be triggered by the release of pyrophosphate, but translocation may not be completed until the binding of the next nucleotide. The protonation state of the polymerases may also help to coordinate phosphodiester bond formation with translocation. In yeast RNA polymerase II, a multi-subunit polymerase structurally distinct from RdRps, it has been proposed that His1085 on the “trigger loop” acts as a general acid to protonate the β-phosphate of the incoming nucleotide to generate a better pyrophosphate-leaving group [31]. The release of pyrophosphate and/or change in the protonation state of His1085 may lead to conformational changes that drive translocation. Indeed, conformational changes in the trigger loop can induce further structural changes to the “bridgehelix”, which is important for translocation [31]. In the A-family DNA polymerases, the general acid is an absolutely conserved Lys on the O-helix [32]. Again, changes in the protonation state of the general acid may couple to structural changes in the O-helix important for translocation. The general acid in RdRps has been proposed to be an absolutely conserved Lys in the motif-D loop [32]. It was previously demonstrated that the conformational state of PV RdRp depends on the protonation state of the motif-D Lys [33], and a long-range interaction network that would be able to communicate structural dynamic changes from motif-D to motif-B has been proposed [34]. Molecular dynamic simulations have also suggested that motions in the motif-D loop are coupled to fluctuations in motif-A and other regions in the active site [35], which might be important for governing the motif-B conformational state. The function of the general acid is closely tied to translocation, considering that amino acid substitutions at the general acid substantially reduce processivity [32].
Viral RNA Polymerase Translocation
Other biophysical studies will likely be required to delineate the sequence of events in RdRp translocation. Single-molecule studies have proven especially revealing in other polymerases and generally support the “passive” model of translocation [36–38]. NMR studies may also reveal the capacity for the RdRp to fluctuate between pre-translocated and post-translocated states [39]. Further X-ray crystallography studies in the presence of excess pyrophosphate and 3′ deoxy RNA may reveal other structural states important in the translocation process. The site mutants studied by Sholders and Peersen should serve as important tools and starting points in the further unraveling of the translocation mechanism and its relationship to RNA replication speed and fidelity.
Acknowledgements I thank Drs. Jamie Arnold and Craig Cameron for their insightful comments.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercialShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
References [1] Ng KK, Arnold JJ, Cameron CE. Structure–function relationships among RNA-dependent RNA polymerases. Curr Top Microbiol Immunol 2008;320:137–56. [2] Waheed Y, Bhatti A, Ashraf M. RNA dependent RNA polymerase of HCV: a potential target for the development of antiviral drugs. Infect Genet Evol 2013;14:247–57. [3] Malet H, Masse N, Selisko B, Romette JL, Alvarez K, Guillemot JC, et al. The flavivirus polymerase as a target for drug discovery. Antiviral Res 2008;80:23–35. [4] Vignuzzi M, Stone JK, Andino R. Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications. Virus Res 2005;107: 173–81. [5] Ollis DL, Brick P, Hamlin R, Xuong NG, Steitz TA. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 1985;313:762–6. [6] Steitz TA. A mechanism for all polymerases. Nature 1998;391:231–2. [7] Berdis AJ. Mechanisms of DNA polymerases. Chem Rev 2009;109:2862–79. [8] Steitz TA. Visualizing polynucleotide polymerase machines at work. EMBO J 2006;25:3458–68. [9] Ferrer-Orta C, Agudo R, Domingo E, Verdaguer N. Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr Opin Struct Biol 2009;19:752–8.
1375 [10] Lescar J, Canard B. RNA-dependent RNA polymerases from flaviviruses and Picornaviridae. Curr Opin Struct Biol 2009;19:759–67. [11] Cheetham GM, Jeruzalmi D, Steitz TA. Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature 1999;399:80–3. [12] Yin YW, Steitz TA. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 2002;298:1387–95. [13] Yin YW, Steitz TA. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 2004;116:393–404. [14] Li Y, Korolev S, Waksman G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J 1998;17:7514–25. [15] Golosov AA, Warren JJ, Beese LS, Karplus M. The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis. Structure 2010;18:83–93. [16] Hansen JL, Long AM, Schultz SC. Structure of the RNAdependent RNA polymerase of poliovirus. Structure 1997;5:1109–22. [17] O'Reilly EK, Kao CC. Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology 1998;252:287–303. [18] Garriga D, Ferrer-Orta C, Querol-Audi J, Oliva B, Verdaguer N. Role of motif B loop in allosteric regulation of RNA-dependent RNA polymerization activity. J Mol Biol 2013;425:2279–87. [19] McDonald SM, Tao YJ, Patton JT. The ins and outs of fourtunneled Reoviridae RNA-dependent RNA polymerases. Curr Opin Struct Biol 2009;19:775–82. [20] Gong P, Kortus MG, Nix JC, Davis RE, Peersen OB. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts. PLoS One 2013;8:e60272. [21] Kortus MG, Kempf BJ, Haworth KG, Barton DJ, Peersen OB. A template RNA entry channel in the fingers domain of the poliovirus polymerase. J Mol Biol 2012;417:263–78. [22] Gong P, Peersen OB. Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A 2010;107:22505–10. [23] Svetlov V, Nudler E. Macromolecular micromovements: how RNA polymerase translocates. Curr Opin Struct Biol 2009;19:701–7. [24] Kireeva M, Kashlev M, Burton ZF. Translocation by multisubunit RNA polymerases. Biochim Biophys Acta 2010;1799:389–401. [25] Temiakov D, Patlan V, Anikin M, McAllister WT, Yokoyama S, Vassylyev DG. Structural basis for substrate selection by t7 RNA polymerase. Cell 2004;116:381–91. [26] Wang HY, Elston T, Mogilner A, Oster G. Force generation in RNA polymerase. Biophys J 1998;74:1186–202. [27] Landick R. Active-site dynamics in RNA polymerases. Cell 2004;116:351–3. [28] Guajardo R, Sousa R. A model for the mechanism of polymerase translocation. J Mol Biol 1997;265:8–19. [29] Bar-Nahum G, Epshtein V, Ruckenstein AE, Rafikov R, Mustaev A, Nudler E. A ratchet mechanism of transcription elongation and its control. Cell 2005;120:183–93. [30] von Hippel PH. An integrated model of the transcription complex in elongation, termination, and editing. Science 1998;281:660–5.
1376 [31] Wang D, Bushnell DA, Westover KD, Kaplan CD, Kornberg RD. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 2006;127:941–54. [32] Castro C, Smidansky ED, Arnold JJ, Maksimchuk KR, Moustafa I, Uchida A, et al. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat Struct Mol Biol 2009;16:212–8. [33] Yang X, Smidansky ED, Maksimchuk KR, Lum D, Welch JL, Arnold JJ, et al. Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition. Structure 2012;29:1519–27. [34] Yang X, Welch JL, Arnold JJ, Boehr DD. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance. Biochemistry 2010;49:9361–71. [35] Moustafa IM, Shen H, Morton B, Colina CM, Cameron CE. Molecular dynamics simulations of viral RNA-dependent
Viral RNA Polymerase Translocation
[36]
[37]
[38]
[39]
RNA polymerases link conserved and correlated motions of functional elements to fidelity. J Mol Biol 2011;410:159–81. Dahl JM, Mai AH, Cherf GM, Jetha NN, Garalde DR, Marziali A, et al. Direct observation of translocation in individual DNA polymerase complexes. J Biol Chem 2012;287:13407–21. Lieberman KR, Dahl JM, Mai AH, Akeson M, Wang H. Dynamics of the translocation step measured in individual DNA polymerase complexes. J Am Chem Soc 2012;134:18816–23. Dangkulwanich M, Ishibashi T, Liu S, Kireeva ML, Lubkowska L, Kashlev M, et al. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. Elife 2013;2:e00971. Loria JP, Berlow RB, Watt ED. Characterization of enzyme motions by solution NMR relaxation dispersion. Acc Chem Res 2008;41:214–21.