skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

Abstract

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation.

Authors:
 [1];  [2];  [3];  [4];  [4];  [4];  [5]
  1. Jason L Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, United States, Department of Chemistry, University of California, Berkeley, Berkeley, United States
  2. Jason L Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, United States, California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States
  3. Jason L Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, United States, Department of Physics, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
  4. Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research–National Cancer Institute, Frederick, United States
  5. Jason L Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, United States, Department of Chemistry, University of California, Berkeley, Berkeley, United States, California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States, Department of Physics, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1197625
Alternate Identifier(s):
OSTI ID: 1197626
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
eLife
Additional Journal Information:
Journal Name: eLife Journal Volume: 2; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Country of Publication:
United States
Language:
English

Citation Formats

Dangkulwanich, Manchuta, Ishibashi, Toyotaka, Liu, Shixin, Kireeva, Maria L., Lubkowska, Lucyna, Kashlev, Mikhail, and Bustamante, Carlos J. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. United States: N. p., 2013. Web. doi:10.7554/eLife.00971.
Dangkulwanich, Manchuta, Ishibashi, Toyotaka, Liu, Shixin, Kireeva, Maria L., Lubkowska, Lucyna, Kashlev, Mikhail, & Bustamante, Carlos J. Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism. United States. doi:10.7554/eLife.00971.
Dangkulwanich, Manchuta, Ishibashi, Toyotaka, Liu, Shixin, Kireeva, Maria L., Lubkowska, Lucyna, Kashlev, Mikhail, and Bustamante, Carlos J. Tue . "Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism". United States. doi:10.7554/eLife.00971.
@article{osti_1197625,
title = {Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism},
author = {Dangkulwanich, Manchuta and Ishibashi, Toyotaka and Liu, Shixin and Kireeva, Maria L. and Lubkowska, Lucyna and Kashlev, Mikhail and Bustamante, Carlos J.},
abstractNote = {During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation.},
doi = {10.7554/eLife.00971},
journal = {eLife},
number = ,
volume = 2,
place = {United States},
year = {2013},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.7554/eLife.00971

Citation Metrics:
Cited by: 41 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Structural Basis of Transcription: Backtracked RNA Polymerase II at 3.4 Angstrom Resolution
journal, May 2009


Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner
journal, March 2007

  • Galburt, Eric A.; Grill, Stephan W.; Wiedmann, Anna
  • Nature, Vol. 446, Issue 7137
  • DOI: 10.1038/nature05701

A Unified Model of Transcription Elongation: What Have We Learned from Single-Molecule Experiments?
journal, March 2011

  • Ó Maoiléidigh, Dáibhid; Tadigotla, Vasisht R.; Nudler, Evgeny
  • Biophysical Journal, Vol. 100, Issue 5
  • DOI: 10.1016/j.bpj.2010.12.3734

Structural basis of initial RNA polymerase II transcription: Initial RNA Pol II transcription
journal, November 2011

  • Cheung, Alan C. M.; Sainsbury, Sarah; Cramer, Patrick
  • The EMBO Journal, Vol. 30, Issue 23
  • DOI: 10.1038/emboj.2011.396

RNA Polymerase Switches between Inactivated and Activated States By Translocating Back and Forth along the DNA and the RNA
journal, June 1997

  • Komissarova, Natalia; Kashlev, Mikhail
  • Journal of Biological Chemistry, Vol. 272, Issue 24
  • DOI: 10.1074/jbc.272.24.15329

Thermal Probing of E. coli RNA Polymerase Off-Pathway Mechanisms
journal, October 2008

  • Mejia, Yara X.; Mao, Hanbin; Forde, Nancy R.
  • Journal of Molecular Biology, Vol. 382, Issue 3
  • DOI: 10.1016/j.jmb.2008.06.079

The Single-Nucleotide Addition Cycle in Transcription: a Biophysical and Biochemical Perspective
journal, June 1992


The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes
journal, November 2011

  • Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney
  • Nature Structural & Molecular Biology, Vol. 18, Issue 12
  • DOI: 10.1038/nsmb.2164

Entropic elasticity of lambda-phage DNA
journal, September 1994


RNA polymerase stalls in a post-translocated register and can hyper-translocate
journal, September 2012

  • Nedialkov, Yuri A.; Nudler, Evgeny; Burton, Zachary F.
  • Transcription, Vol. 3, Issue 5
  • DOI: 10.4161/trns.22307

Intrinsic Translocation Barrier as an Initial Step in Pausing by RNA Polymerase II
journal, February 2013

  • Imashimizu, Masahiko; Kireeva, Maria L.; Lubkowska, Lucyna
  • Journal of Molecular Biology, Vol. 425, Issue 4
  • DOI: 10.1016/j.jmb.2012.12.002

Active site opening and closure control translocation of multisubunit RNA polymerase
journal, May 2012

  • Malinen, Anssi M.; Turtola, Matti; Parthiban, Marimuthu
  • Nucleic Acids Research, Vol. 40, Issue 15
  • DOI: 10.1093/nar/gks383

Applied Force Provides Insight into Transcriptional Pausing and Its Modulation by Transcription Factor NusA
journal, November 2011


Bridge helix and trigger loop perturbations generate superactive RNA polymerases
journal, January 2008

  • Tan, Lin; Wiesler, Simone; Trzaska, Dominika
  • Journal of Biology, Vol. 7, Issue 10
  • DOI: 10.1186/jbiol98

Thinking quantitatively about transcriptional regulation
journal, March 2005

  • Greive, Sandra J.; von Hippel, Peter H.
  • Nature Reviews Molecular Cell Biology, Vol. 6, Issue 3
  • DOI: 10.1038/nrm1588

The RNA Polymerase II Trigger Loop Functions in Substrate Selection and Is Directly Targeted by α-Amanitin
journal, June 2008


Direct observation of base-pair stepping by RNA polymerase
journal, November 2005

  • Abbondanzieri, Elio A.; Greenleaf, William J.; Shaevitz, Joshua W.
  • Nature, Vol. 438, Issue 7067
  • DOI: 10.1038/nature04268

Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation: Temperature dependence of thermodynamic properties
journal, June 2002


Thermodynamic and kinetic modeling of transcriptional pausing
journal, March 2006

  • Tadigotla, V. R.; Maoileidigh, D. O.; Sengupta, A. M.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 12
  • DOI: 10.1073/pnas.0600508103

Structural Basis of Transcription Inhibition by Antibiotic Streptolydigin
journal, September 2005


Sequence-dependent Kinetic Model for Transcription Elongation by RNA Polymerase
journal, November 2004

  • Bai, Lu; Shundrovsky, Alla; Wang, Michelle D.
  • Journal of Molecular Biology, Vol. 344, Issue 2
  • DOI: 10.1016/j.jmb.2004.08.107

The Origin of Short Transcriptional Pauses
journal, March 2009


A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli
journal, January 1991

  • Yager, Thomas D.; Von Hippel, Peter H.
  • Biochemistry, Vol. 30, Issue 4
  • DOI: 10.1021/bi00218a032

Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded
journal, March 1997

  • Komissarova, N.; Kashlev, M.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 5
  • DOI: 10.1073/pnas.94.5.1755

Measurement of histone–DNA interaction free energy in nucleosomes
journal, May 2004


Rapid spontaneous accessibility of nucleosomal DNA
journal, December 2004

  • Li, Gu; Levitus, Marcia; Bustamante, Carlos
  • Nature Structural & Molecular Biology, Vol. 12, Issue 1
  • DOI: 10.1038/nsmb869

Unwrapping of Nucleosomal DNA Ends: A Multiscale Molecular Dynamics Study
journal, February 2012


Mechanism of Translesion Transcription by RNA Polymerase II and Its Role in Cellular Resistance to DNA Damage
journal, April 2012


Nucleosome Immobilization Strategies for Single-Pair FRET Microscopy
journal, October 2008

  • Koopmans, Wiepke J. A.; Schmidt, Thomas; van Noort, John
  • ChemPhysChem, Vol. 9, Issue 14
  • DOI: 10.1002/cphc.200800370

Nucleosomal Elements that Control the Topography of the Barrier to Transcription
journal, November 2012


RNA Polymerase Backtracking in Gene Regulation and Genome Instability
journal, June 2012


Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase
journal, August 2002

  • Forde, N. R.; Izhaky, D.; Woodcock, G. R.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 18
  • DOI: 10.1073/pnas.142417799

Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation
journal, June 2008


Structural Basis of Transcription: Role of the Trigger Loop in Substrate Specificity and Catalysis
journal, December 2006


Eukaryotic transcriptional dynamics: from single molecules to cell populations
journal, July 2013

  • Coulon, Antoine; Chow, Carson C.; Singer, Robert H.
  • Nature Reviews Genetics, Vol. 14, Issue 8
  • DOI: 10.1038/nrg3484

New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning
journal, February 1998


Reaction pathways in transcript elongation
journal, December 2002


Improved Thermodynamic Parameters and Helix Initiation Factor to Predict Stability of DNA Duplexes
journal, November 1996

  • Sugimoto, N.; Nakano, S. -i.; Yoneyama, M.
  • Nucleic Acids Research, Vol. 24, Issue 22
  • DOI: 10.1093/nar/24.22.4501

Partition analysis and concept of net rate constants as tools in enzyme kinetics
journal, July 1975


Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules
journal, June 2006


Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation
journal, June 2008

  • Brueckner, Florian; Cramer, Patrick
  • Nature Structural & Molecular Biology, Vol. 15, Issue 8
  • DOI: 10.1038/nsmb.1458

Mechanism of sequence-specific pausing of bacterial RNA polymerase
journal, April 2009

  • Kireeva, M. L.; Kashlev, M.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 22
  • DOI: 10.1073/pnas.0900407106

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases
journal, May 2012

  • Zamft, B.; Bintu, L.; Ishibashi, T.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 23
  • DOI: 10.1073/pnas.1205063109

Architecture of the RNA Polymerase II-TFIIS Complex and Implications for mRNA Cleavage
journal, August 2003


Allosteric Binding of Nucleoside Triphosphates to RNA Polymerase Regulates Transcription Elongation
journal, July 2001


Conformational Coupling in DNA Polymerase Fidelity
journal, June 1993


A model for the mechanism of polymerase translocation 1 1Edited by A. R. Fersht
journal, January 1997


Nucleosomal Fluctuations Govern the Transcription Dynamics of RNA Polymerase II
journal, July 2009


The RNA–DNA Hybrid Maintains the Register of Transcription by Preventing Backtracking of RNA Polymerase
journal, April 1997


NTP-driven Translocation by Human RNA Polymerase II
journal, March 2003

  • Nedialkov, Yuri A.; Gong, Xue Q.; Hovde, Stacy L.
  • Journal of Biological Chemistry, Vol. 278, Issue 20
  • DOI: 10.1074/jbc.M301103200

Structural basis for substrate loading in bacterial RNA polymerase
journal, June 2007

  • Vassylyev, Dmitry G.; Vassylyeva, Marina N.; Zhang, Jinwei
  • Nature, Vol. 448, Issue 7150
  • DOI: 10.1038/nature05931

Single-Molecule Studies of RNA Polymerase: Motoring Along
journal, June 2008


Mechanochemical Kinetics of Transcription Elongation
journal, February 2007


Structural Basis of Transcriptional Pausing in Bacteria
journal, January 2013


Translocation by multi-subunit RNA polymerases
journal, May 2010

  • Kireeva, Maria; Kashlev, Mikhail; Burton, Zachary F.
  • Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol. 1799, Issue 5-6
  • DOI: 10.1016/j.bbagrm.2010.01.007

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II
journal, April 2012

  • Larson, M. H.; Zhou, J.; Kaplan, C. D.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 17
  • DOI: 10.1073/pnas.1200939109

Structural Basis of Transcription
journal, November 2004


Nucleosomes Can Form a Polar Barrier to Transcript Elongation by RNA Polymerase II
journal, November 2006


A Ratchet Mechanism of Transcription Elongation and Its Control
journal, January 2005


Nature of the Nucleosomal Barrier to RNA Polymerase II
journal, April 2005


A Central Role of the RNA Polymerase Trigger Loop in Active-Site Rearrangement during Transcriptional Pausing
journal, August 2007


Structural basis of RNA polymerase II backtracking, arrest and reactivation
journal, February 2011


Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals
journal, June 2000

  • Artsimovitch, I.; Landick, R.
  • Proceedings of the National Academy of Sciences, Vol. 97, Issue 13
  • DOI: 10.1073/pnas.97.13.7090

DNA Translocation and Loop Formation Mechanism of Chromatin Remodeling by SWI/SNF and RSC
journal, November 2006


Dynamic Error Correction and Regulation of Downstream Bubble Opening by Human RNA Polymerase II
journal, May 2005


The regulatory roles and mechanism of transcriptional pausing
journal, October 2006


Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil
journal, March 2006


Structural Basis of Transcription: Mismatch-Specific Fidelity Mechanisms and Paused RNA Polymerase II with Frayed RNA
journal, June 2009


The Structural Mechanism of Translocation and Helicase Activity in T7 RNA Polymerase
journal, February 2004