期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 43, 页码 E6562-E6571出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1605038113
关键词
transcription; pausing; backtracking; RNAP; RNA polymerase
资金
- CNRS
- University of Paris Diderot
- Programme Equipe Labellisees of the French Ligue Contre le Cancer
- National Institutes of Health [GM069709, GM095904]
- National Science Foundation [MCB-1244175]
- Direct For Biological Sciences [1244098] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience [1244098] Funding Source: National Science Foundation
Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2-or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.
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