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Optical tweezers studies of transcription by eukaryotic RNA polymerases

Ana Lisica
  • BIOTEC, Technical University Dresden, Tatzberg 47/49, D-01307 Dresden, Germany; and Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, D-01307 Dresden, Germany
/ Stephan W. Grill
  • Corresponding author
  • BIOTEC, Technical University Dresden, Tatzberg 47/49, D-01307 Dresden, Germany; and Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, D-01307 Dresden, Germany
  • Email:
Published Online: 2017-02-21 | DOI: https://doi.org/10.1515/bmc-2016-0028


Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

Keywords: optical tweezers; RNA polymerases; single-molecule techniques; transcription


  • 1.

    Cramer P, Armache KJ, Baumli S, Benkert S, Brueckner F, Buchen C, Damsma GE, Dengl S, Geiger SR, Jasiak AJ, Jawhari A. Structure of eukaryotic RNA polymerases. Annu Rev Biophys 2008; 37: 337–52.

  • 2.

    Jonkers I, Lis JT. Getting up to speed with transcription elongation by RNA polymerase II. Nat Rev Mol Cell Biol 2015; 16: 167–77.

  • 3.

    Darzacq X, Yao J, Larson DR, Causse SZ, Bosanac L, De Turris V, Ruda VM, Lionnet T, Zenklusen D, Guglielmi B, Tjian R. Imaging transcription in living cells. Annu Rev Biophys 2009; 38: 173.

  • 4.

    Bustamante C, Cheng W, Mejia YX. Revisiting the central dogma one molecule at a time. Cell 2011; 144: 480–97.

  • 5.

    Larson MH, Landick R, Block SM. Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes. Mol Cell 2011; 41: 249–62.

  • 6.

    Galburt EA, Grill SW, Bustamante C. Single molecule transcription elongation. Methods 2009; 48: 323–32.

  • 7.

    Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM. Direct observation of base-pair stepping by RNA polymerase. Nature 2005; 438: 460–5.

  • 8.

    Dalal RV, Larson MH, Neuman KC, Gelles J, Landick R, Block SM. Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing. Mol Cell 2006; 23: 231–9.

  • 9.

    Schweikhard V, Meng C, Murakami K, Kaplan CD, Kornberg RD, Block SM. Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms. Proc Natl Acad Sci USA 2014; 111: 6642–7.

  • 10.

    Fazal FM, Meng CA, Murakami K, Kornberg RD, Block SM. Real-time observation of the initiation of RNA polymerase II transcription. Nature 2015; 525: 274–7.

  • 11.

    Lisica A, Engel C, Jahnel M, Roldán É, Galburt EA, Cramer P, Grill SW. Mechanisms of backtrack recovery by RNA polymerases I and II. Proc Natl Acad Sci USA 2016; 113: 2946–51.

  • 12.

    Fazal FM, Block SM. Optical tweezers study life under tension. Nat Photonics 2011; 5: 318–21.

  • 13.

    Ashkin A. Acceleration and trapping of particles by radiation pressure. Phys Rev Lett 1970; 24: 156.

  • 14.

    Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S. Observation of a single-beam gradient force optical trap for dielectric particles. Optics Lett 1986; 11: 288–90.

  • 15.

    Neuman KC, Block SM. Optical trapping. Rev Sci Instr 2004; 75: 2787–809.

  • 16.

    Berg-Sørensen K, Flyvbjerg H. Power spectrum analysis for optical tweezers. Rev Sci Instr 2004; 75: 594–612.

  • 17.

    Van Mameren J, Peterman EJ, Wuite GJ. See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins. Nucleic Acids Res 2008; 36: 4381–9.

  • 18.

    Smith SB, Cui Y, Bustamente C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 1996; 271: 795.

  • 19.

    Wang MD, Yin H, Landick R, Gelles J, Block SM. Stretching DNA with optical tweezers. Biophys J 1997; 72: 1335.

  • 20.

    Wuite GJ, Smith SB, Young M, Keller D, Bustamante C. Single-molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 2000; 404: 103–6.

  • 21.

    Dangkulwanich M, Ishibashi T, Liu S, Kireeva ML, Lubkowska L, Kashlev M, Bustamante CJ. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. eLife 2013; 2: e00971.

  • 22.

    Hodges C, Bintu L, Lubkowska L, Kashlev M, Bustamante C. Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II. Science 2009; 325: 626–8.

  • 23.

    Bintu L, Ishibashi T, Dangkulwanich M, Wu YY, Lubkowska L, Kashlev M, Bustamante C. Nucleosomal elements that control the topography of the barrier to transcription. Cell 2012; 151: 738–49.

  • 24.

    Fitz V, Shin J, Ehrlich C, Farnung L, Cramer P, Zaburdaev V, Grill SW. Nucleosomal arrangement affects single-molecule transcription dynamics. Proc Natl Acad Sci USA 2016; 45: 12733–8.

  • 25.

    Conaway RC, Conaway JW. General initiation factors for RNA polymerase II. Annu Rev Biochem 1993; 62: 161–90.

  • 26.

    Kapanidis AN, Margeat E, Ho SO, Kortkhonjia E, Weiss S, Ebright RH. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 2006; 314: 1144–7.

  • 27.

    Revyakin A, Liu C, Ebright RH, Strick TR. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 2006; 314: 1139–43.

  • 28.

    Kapanidis AN, Margeat E, Laurence TA, Doose S, Ho SO, Mukhopadhyay J, Kortkhonjia E, Mekler V, Ebright RH, Weiss S. Retention of transcription initiation factor s 70 in transcription elongation: single-molecule analysis. Mol Cell 2005; 20: 347–56.

  • 29.

    Chakraborty A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R, Nixon BT. Opening and closing of the bacterial RNA polymerase clamp. Science 2012; 337: 591–5.

  • 30.

    Duchi D, Bauer DL, Fernandez L, Evans G, Robb N, Hwang LC, Gryte K, Tomescu A, Zawadzki P, Morichaud Z, Brodolin K. RNA polymerase pausing during initial transcription. Mol Cell 2016; 63: 939–50.

  • 31.

    Yin H, Wang MD, Svoboda K, Landick R. Transcription against an applied force. Science 1995; 270: 1653.

  • 32.

    Wang MD, Schnitzer MJ, Yin H, Landick R, Gelles J, Block SM. Force and velocity measured for single molecules of RNA polymerase. Science 1998; 282: 902–7.

  • 33.

    Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 2011; 332: 475–8.

  • 34.

    Zamft B, Bintu L, Ishibashi T, Bustamante C. Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases. Proc Natl Acad Sci USA 2012; 109: 8948–53.

  • 35.

    Ishibashi T, Dangkulwanich M, Coello Y, Lionberger TA, Lubkowska L, Ponticelli AS, Kashlev M, Bustamante C. Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics. Proc Natl Acad Sci USA 2014; 111: 3419–24.

  • 36.

    Engel C, Sainsbury S, Cheung AC, Kostrewa D, Cramer P. RNA polymerase I structure and transcription regulation. Nature 2013; 502: 650–5.

  • 37.

    Fernández-Tornero C, Moreno-Morcillo M, Rashid UJ, Taylor NM, Ruiz FM, Gruene T, Legrand P, Steuerwald U, Müller CW. Crystal structure of the 14-subunit RNA polymerase I. Nature 2013; 502: 644–9.

  • 38.

    Kuhn CD, Geiger SR, Baumli S, Gartmann M, Gerber J, Jennebach S, Mielke T, Tschochner H, Beckmann R, Cramer P. Functional architecture of RNA polymerase I. Cell 2007; 131: 1260–72.

  • 39.

    Erie DA, Yager TD, Von Hippel PH. The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective. Annu Rev Biophys Biomol Struct 1992; 21: 379–415.

  • 40.

    Zhou J, Schweikhard V, Block SM. Single-molecule studies of RNAPII elongation. Biochim Biophys Acta Gene Regul Mech 2013; 1829: 29–38.

  • 41.

    Guajardo R, Sousa R. A model for the mechanism of polymerase translocation. J Mol Biol 1997; 265: 8–19.

  • 42.

    Komissarova N, Kashlev M. RNA polymerase switches between inactivated and activated states by translocating back and forth along the DNA and the RNA. J Biol Chem 1997; 272: 15329–38.

  • 43.

    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.

  • 44.

    Larson MH, Zhou J, Kaplan CD, Palangat M, Kornberg RD, Landick R, Block SM. Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II. Proc Natl Acad Sci USA 2012; 109: 6555–60.

  • 45.

    Muse GW, Gilchrist DA, Nechaev S, Shah R, Parker JS, Grissom SF, Zeitlinger J, Adelman K. RNA polymerase is poised for activation across the genome. Nat Genet 2007; 39: 1507–11.

  • 46.

    Oesterreich FC, Preibisch S, Neugebauer KM. Global analysis of nascent RNA reveals transcriptional pausing in terminal exons. Mol Cell 2010; 40: 571–81.

  • 47.

    Alexander RD, Innocente SA, Barrass JD, Beggs JD. Splicing-dependent RNA polymerase pausing in yeast. Mol Cell 2010; 40: 582–93.

  • 48.

    Nudler E, Mustaev A, Goldfarb A, Lukhtanov E. The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 1997; 89: 33–41.

  • 49.

    Komissarova N, Kashlev M. Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded. Proc Natl Acad Sci USA 1997; 94: 1755–60.

  • 50.

    Kettenberger H, Armache KJ, Cramer P. Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 2003; 114: 347–57.

  • 51.

    Wang D, Bushnell DA, Huang X, Westover KD, Levitt M, Kornberg RD. Structural basis of transcription: backtracked RNA polymerase II at 3.4 Å resolution. Science 2009; 324: 1203–6.

  • 52.

    Cheung AC, Cramer P. Structural basis of RNA polymerase II backtracking, arrest and reactivation. Nature 2011; 471: 249–53.

  • 53.

    Churchman LS, Weissman JS. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 2011; 469: 368–73.

  • 54.

    Mayer A, Di Iulio J, Maleri S, Eser U, Vierstra J, Reynolds A, Sandstrom R, Stamatoyannopoulos JA, Churchman LS. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell 2015; 161: 541–54.

  • 55.

    Shaevitz JW, Abbondanzieri EA, Landick R, Block SM. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 2003; 426: 684–7.

  • 56.

    Galburt EA, Grill SW, Wiedmann A, Lubkowska L, Choy J, Nogales E, Kashlev M, Bustamante C. Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. Nature 2007; 446: 820–3.

  • 57.

    Erie DA, Hajiseyedjavadi O, Young MC, Von Hippel PH. Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. Science 1993; 262: 867–73.

  • 58.

    Thomas MJ, Platas AA, Hawley DK. Transcriptional fidelity and proofreading by RNA polymerase II. Cell 1998; 93: 627–37.

  • 59.

    Sydow JF, Brueckner F, Cheung AC, Damsma GE, Dengl S, Lehmann E, Vassylyev D, Cramer P. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol Cell 2009; 34: 710–21.

  • 60.

    Depken M, Parrondo JM, Grill SW. Intermittent transcription dynamics for the rapid production of long transcripts of high fidelity. Cell Rep 2013; 5: 521–30.

  • 61.

    Depken M, Galburt EA, Grill SW. The origin of short transcriptional pauses. Biophys J 2009; 96: 2189–93.

  • 62.

    Walmacq C, Kireeva ML, Irvin J, Nedialkov Y, Lubkowska L, Malagon F, Strathern JN, Kashlev M. Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II. J Biol Chem 2009; 284: 19601–12.

  • 63.

    Chédin S, Riva M, Schultz P, Sentenac A, Carles C. The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination. Genes Dev 1998; 12: 3857–71.

  • 64.

    Izban MG, Luse DS. The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'?5'direction in the presence of elongation factor SII. Genes Dev 1992; 6: 1342–56.

  • 65.

    Fish RN, Kane CM. Promoting elongation with transcript cleavage stimulatory factors. Biochim Biophys Acta Gene Struct Expr 2002; 1577: 287–307.

  • 66.

    Ruan W, Lehmann E, Thomm M, Kostrewa D, Cramer P. Evolution of two modes of intrinsic RNA polymerase transcript cleavage. J Biol Chem 2011; 286: 18701–7.

  • 67.

    Klopper AV, Bois JS, Grill SW. Influence of secondary structure on recovery from pauses during early stages of RNA transcription. Phys Rev E 2010; 81: 030904.

  • 68.

    Neri I, Roldán É, Jülicher F. Statistics of infima and stopping times of entropy production and applications to active molecular processes. Pays Rev X 2017; (in press). arXiv:1604.04159.

  • 69.

    Palangat M, Landick R. Roles of RNA: DNA hybrid stability, RNA structure, and active site conformation in pausing by human RNA polymerase II. J Mol Biol 2001; 311: 265–82.

  • 70.

    Kraeva RI, Krastev DB, Roguev A, Ivanova A, Nedelcheva-Veleva MN, Stoynov SS. Stability of mRNA/DNA and DNA/DNA duplexes affects mRNA transcription. PLoS One 2007; 2: e290. [Crossref]

  • 71.

    Nedelcheva-Veleva MN, Sarov M, Yanakiev I, Mihailovska E, Ivanov MP, Panova GC, Stoynov SS. The thermodynamic patterns of eukaryotic genes suggest a mechanism for intron-exon recognition. Nat Commun 2013; 4: 2101.

  • 72.

    Roldán É, Lisica A, Sánchez-Taltavull D, Grill SW. Stochastic resetting in backtrack recovery by RNA polymerases. Phys Rev E 2016; 93: 062411.

  • 73.

    Neuman KC, Abbondanzieri EA, Landick R, Gelles J, Block SM. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 2003; 115: 437–47.

  • 74.

    Herbert KM, La Porta A, Wong BJ, Mooney RA, Neuman KC, Landick R, Block SM. Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 2006; 125: 1083–94.

  • 75.

    Izban MG, Luse DS. Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. Genes Dev 1991; 5: 683–96.

  • 76.

    Kireeva ML, Walter W, Tchernajenko V, Bondarenko V, Kashlev M, Studitsky VM. Nucleosome remodeling induced by RNA polymerase II: loss of the H2A/H2B dimer during transcription. Mol Cell 2002; 9: 541–52.

  • 77.

    Bondarenko VA, Steele LM, Újvári A, Gaykalova DA, Kulaeva OI, Polikanov YS, Luse DS, Studitsky VM. Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol Cell 2006; 24: 469–79.

  • 78.

    Izban MG, Luse DS. Factor-stimulated RNA polymerase II transcribes at physiological elongation rates on naked DNA but very poorly on chromatin templates. J Biol Chem 1992; 267: 13647–55.

  • 79.

    Cojocaru M, Jeronimo C, Forget D, Bouchard A, Bergeron D, Côte P, Poirier GG, Greenblatt J, Coulombe B. Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription. Biochem J 2008; 409: 139–47.

  • 80.

    Nudler E, Gottesman ME. Transcription termination and anti-termination in E. coli. Genes Cells 2002; 7: 755–68.

  • 81.

    Richard P, Manley JL. Transcription termination by nuclear RNA polymerases. Genes Dev 2009; 23: 1247–69.

  • 82.

    Kuehner JN, Pearson EL, Moore C. Unravelling the means to an end: RNA polymerase II transcription termination. Nat Rev Mol Cell Biol 2011; 12: 283–94.

  • 83.

    Russell J, Zomerdijk JC. RNA-polymerase-I-directed rDNA transcription, life and works. Trends Biochem Sci 2005; 30: 87–96.

  • 84.

    Nielsen S, Yuzenkova Y, Zenkin N. Mechanism of eukaryotic RNA polymerase III transcription termination. Science 2013; 340: 1577–80.

  • 85.

    Larson MH, Greenleaf WJ, Landick R, Block SM. Applied force reveals mechanistic and energetic details of transcription termination. Cell 2008; 132: 971–82.

  • 86.

    Koslover DJ, Fazal FM, Mooney RA, Landick R, Block SM. Binding and translocation of termination factor rho studied at the single-molecule level. J Mol Biol 2012; 423: 664–76.

  • 87.

    Gaspard P. Template-directed copolymerization, random walks along disordered tracks, and fractals. Phys Rev Lett 2016; 117: 238101.

  • 88.

    van Mameren J, Modesti M, Kanaar R, Wyman C, Wuite GJ, Peterman EJ. Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers. Biophys J 2006; 91: L78–80.

  • 89.

    van Mameren J, Gross P, Farge G, Hooijman P, Modesti M, Falkenberg M, Wuite GJ, Peterman EJ. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc Natl Acad Sci USA 2009; 106: 18231–6.

  • 90.

    Gross P, Laurens N, Oddershede LB, Bockelmann U, Peterman EJ, Wuite GJ. Quantifying how DNA stretches, melts and changes twist under tension. Nat Physics 2011; 7: 731–6.

  • 91.

    La Porta A, Wang MD. Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys Rev Lett 2004; 92: 190801.

  • 92.

    Ma J, Bai L, Wang MD. Transcription under torsion. Science 2013; 340: 1580–3.

  • 93.

    Sheinin MY, Li M, Soltani M, Luger K, Wang MD. Torque modulates nucleosome stability and facilitates H2A/H2B dimer loss. Nat Commun 2013; 4: 2579.

About the article

aPresent address: London Centre for Nanotechnology, University College London, London WC1H 0AH, UK

Received: 2016-12-01

Accepted: 2017-01-10

Published Online: 2017-02-21

Published in Print: 2017-03-01

Citation Information: Biomolecular Concepts, ISSN (Online) 1868-503X, ISSN (Print) 1868-5021, DOI: https://doi.org/10.1515/bmc-2016-0028. Export Citation

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