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

Author

Listed:
  • Eric A. Galburt

    (Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
    Present address: Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany)

  • Stephan W. Grill

    (Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
    Present address: Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany)

  • Anna Wiedmann

    (Department of Molecular and Cell Biology,)

  • Lucyna Lubkowska

    (NCI Center for Cancer Research, Frederick, Maryland 21702, USA)

  • Jason Choy
  • Eva Nogales

    (Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
    Department of Molecular and Cell Biology,
    Howard Hughes Medical Institute, University of California, Berkeley 94720, USA)

  • Mikhail Kashlev

    (NCI Center for Cancer Research, Frederick, Maryland 21702, USA)

  • Carlos Bustamante

    (Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
    Department of Molecular and Cell Biology,
    Department of Chemistry and,
    Howard Hughes Medical Institute, University of California, Berkeley 94720, USA)

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.

Suggested Citation

  • Eric A. Galburt & Stephan W. Grill & Anna Wiedmann & Lucyna Lubkowska & Jason Choy & Eva Nogales & Mikhail Kashlev & Carlos Bustamante, 2007. "Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner," Nature, Nature, vol. 446(7137), pages 820-823, April.
    • Handle: RePEc:nat:nature:v:446:y:2007:i:7137:d:10.1038_nature05701
    DOI: 10.1038/nature05701
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    Cited by:

    1. Ying Xiong & Weijing Han & Chunhua Xu & Jing Shi & Lisha Wang & Taoli Jin & Qi Jia & Ying Lu & Shuxin Hu & Shuo-Xing Dou & Wei Lin & Terence R. Strick & Shuang Wang & Ming Li, 2024. "Single-molecule reconstruction of eukaryotic factor-dependent transcription termination," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    2. Jordan Douglas & Richard Kingston & Alexei J Drummond, 2020. "Bayesian inference and comparison of stochastic transcription elongation models," PLOS Computational Biology, Public Library of Science, vol. 16(2), pages 1-21, February.

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