1. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
2. Department of Molecular and Cell Biology,
3. Department of Chemistry and,
4. Howard Hughes Medical Institute, University of California, Berkeley 94720, USA
5. NCI Center for Cancer Research, Frederick, Maryland 21702, USA
6. These authors contributed equally to this work.
7. Present address: Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany
8. Deceased.

Correspondence to: Carlos Bustamante^1,2,3,4 Correspondence and requests for materials should be addressed to C.B. (Email: carlos@alice.berkeley.edu).

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 backtracking^{3, 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 RNAP^{7, 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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