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

Eric A. Galburt, Stephan W. Grill, Anna Wiedmann, Lucyna Lubkowska, Jason Choy, Eva Nogales, Mikhail Kashlev and Carlos Bustamante ()
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Eric A. Galburt: Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
Stephan W. Grill: Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
Anna Wiedmann: Department of Molecular and Cell Biology,
Lucyna Lubkowska: NCI Center for Cancer Research, Frederick, Maryland 21702, USA
Eva Nogales: Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
Mikhail Kashlev: NCI Center for Cancer Research, Frederick, Maryland 21702, USA
Carlos Bustamante: Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

Nature, 2007, vol. 446, issue 7137, 820-823

Abstract: 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.

Date: 2007
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DOI: 10.1038/nature05701

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