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Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner

Nature volume 446, pages 820–823 (2007)Cite this article

Abstract

RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human1, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers2. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking3,4,5,6. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 ± 2 pN, only one-third of the force determined for Escherichia coli RNAP7,8. We show that backtrack pause durations follow a t-3/2 power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 ± 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.

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Figure 1: Single-molecule transcription.
Figure 2: Force–velocity analysis.
Figure 3: Backtrack entry and exit.

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Acknowledgements

We thank Y. R. Chemla, W. Cheng, M. Cruse, S. Dumont, N. R. Forde, B. Ibarra, D. Izhaky, C. Kane, S. Kostek, J. Moffit, J. M. R. Parrondo, M. Peris, S. Plyasunov, A. Ruiz and S. B. Smith for experimental assistance and helpful discussions. This research was supported by a DOE grant. E.A.G. was supported by a Jane Coffin Childs Postdoctoral Fellowship. S.W.G. was supported first by an EMBO Long Term Fellowship and subsequently by a Helen Hay Whitney Postdoctoral Fellowship. We dedicate this manuscript to Jason Choy.

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Author notes

  1. Eric A. Galburt & Stephan W. Grill

    Present address: Present address: Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany,

  2. Eric A. Galburt and Stephan W. Grill: These authors contributed equally to this work.

  3. Jason Choy: Deceased.

Authors and Affiliations

  1. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA,

    Eric A. Galburt, Stephan W. Grill, Eva Nogales & Carlos Bustamante

  2. Department of Molecular and Cell Biology,,

    Anna Wiedmann, Eva Nogales & Carlos Bustamante

  3. Department of Chemistry and,,

    Carlos Bustamante

  4. Howard Hughes Medical Institute, University of California, Berkeley 94720, USA,

    Eva Nogales & Carlos Bustamante

  5. NCI Center for Cancer Research, Frederick, Maryland 21702, USA,

    Lucyna Lubkowska & Mikhail Kashlev

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Correspondence to Carlos Bustamante.

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This file contains Supplementary Figures S1- S8, Supplementary Table S1, Supplementary Methods, Supplementary Equations and additional references (PDF 600 kb)

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Galburt, E., Grill, S., Wiedmann, A. et al. Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. Nature 446, 820–823 (2007). https://doi.org/10.1038/nature05701

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