Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner.
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| Title: | Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. |
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| Authors: | Galburt, Eric A., Grill, Stephan W., Wiedmann, Anna, Lubkowska, Lucyna, Choy, Jason, Nogales, Eva, Kashlev, Mikhail, Bustamante, Carlos |
| Source: | Nature. 4/12/2007, Vol. 446 Issue 7137, p820-823. 4p. 3 Graphs. |
| Subjects: | RNA polymerases, Messenger RNA, Eukaryotic cells, Saccharomyces cerevisiae, Enzymes |
| 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 human, 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 barriers. 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. 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. 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. [ABSTRACT FROM AUTHOR] |
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| Database: | Psychology and Behavioral Sciences Collection |
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