Article ; Online: Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.
Proceedings of the National Academy of Sciences of the United States of America
2016 Volume 113, Issue 43, Page(s) E6562–E6571
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 ... ...
| 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. |
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| MeSH term(s) | Base Sequence ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Gene Expression Regulation, Bacterial ; Guanosine Triphosphate/metabolism ; Kinetics ; Nucleic Acid Conformation ; RNA Polymerase II/genetics ; RNA Polymerase II/metabolism ; RNA, Messenger/chemistry ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Sequence Alignment ; Transcription Factors/genetics ; Transcription Factors/metabolism ; Transcription Initiation, Genetic ; Uridine Triphosphate/metabolism |
| Chemical Substances | Escherichia coli Proteins ; GreA protein, E coli ; RNA, Messenger ; Transcription Factors ; Guanosine Triphosphate (86-01-1) ; RNA Polymerase II (EC 2.7.7.-) ; Uridine Triphosphate (UT0S826Z60) |
| Language | English |
| Publishing date | 2016-10-11 |
| Publishing country | United States |
| Document type | Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S. |
| ZDB-ID | 209104-5 |
| ISSN | 1091-6490 ; 0027-8424 |
| ISSN (online) | 1091-6490 |
| ISSN | 0027-8424 |
| DOI | 10.1073/pnas.1605038113 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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