Backtracking determines the force sensitivity of RNAPII in a factor-dependent manner
Abstract
RNA polymerase II (RNAPII) 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 RNAPII 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 RNAPII to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAPII molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5+/-2pN, 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 RNAPII diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAPII pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9+/-3.4pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAPII, allowing it to operate against higher loads.
- Publication:
-
Nature
- Pub Date:
- April 2007
- DOI:
- 10.1038/nature05701
- Bibcode:
- 2007Natur.446..820G