title: Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner.
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.
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Galburt
authors.forename: Stephan W
authors.initials: SW
authors.lastname: Grill
authors.forename: Anna
authors.initials: A
authors.lastname: Wiedmann
authors.forename: Lucyna
authors.initials: L
authors.lastname: Lubkowska
authors.forename: Jason
authors.initials: J
authors.lastname: Choy
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
mesh_terms: D001696:Biomechanical Phenomena; D004926:Escherichia coli / Q000201:enzymology; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000378:metabolism
chemical_list: D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II