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Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.


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.

SUBMITTER: Lerner E 

PROVIDER: S-EPMC5087071 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.

Lerner Eitan E   Chung SangYoon S   Allen Benjamin L BL   Wang Shuang S   Lee Jookyung J   Lu Shijia W SW   Grimaud Logan W LW   Ingargiola Antonino A   Michalet Xavier X   Alhadid Yazan Y   Borukhov Sergei S   Strick Terence R TR   Taatjes Dylan J DJ   Weiss Shimon S  

Proceedings of the National Academy of Sciences of the United States of America 20161011 43


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 endonucleas  ...[more]

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