title: Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms.
abstract: Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.
authors.affiliation: Departments of Biology, Chemistry, Structural Biology, and Applied Physics, Stanford University, Stanford, CA 94305.
authors.forename: Volker
authors.initials: V
authors.lastname: Schweikhard
authors.forename: Cong
authors.initials: C
authors.lastname: Meng
authors.forename: Kenji
authors.initials: K
authors.lastname: Murakami
authors.forename: Craig D
authors.initials: CD
authors.lastname: Kaplan
authors.forename: Roger D
authors.initials: RD
authors.lastname: Kornberg
authors.forename: Steven M
authors.initials: SM
authors.lastname: Block
mesh_terms: D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D052898:Optical Tweezers; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D061805:Transcription Elongation, Genetic; D019473:Transcription Factors, TFII / Q000235:genetics / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004793:Enzyme Reactivators; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D007293:Inosine Triphosphate; D012319:RNA Polymerase II; C056604:transcription factor TFIIF
keywords: Pol II; optical trap; optical tweezers
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