Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Eitan Lernera, Sang Yoon Chung, Benjamin L. Allen, Shuang Wang, Jookyung Lee, Shijia W. Lu, Logan W. Grimaud, Antonino Ingargiola, Xavier Michalet, Yazan Alhadid, Sergei Borukhov, Terence R. Strick, Dylan J. Taatjes, Shimon Weiss

    Research output: Contribution to journalArticlepeer-review

    42 Scopus citations

    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)E6562-E6571
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume113
    Issue number43
    DOIs
    StatePublished - Oct 25 2016

    All Science Journal Classification (ASJC) codes

    • General

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