Article ; Online: On the stability of stalled RNA polymerase and its removal by RapA.
2022 Volume 50, Issue 13, Page(s) 7396–7405
Abstract: Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ... ...
Abstract | Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ATP-dependent fashion, was identified over 50 years ago as an abundant binding partner of RNAP; however, its mechanism of action remains unknown. Here, we use single-molecule magnetic trapping assays to characterize RapA activity and begin to specify its mechanism of action. We first show that stalled RNAP resides on DNA for times on the order of 106 seconds and that increasing positive torque on the DNA reduces this lifetime. Using stalled RNAP as a substrate we show that the RapA protein stimulates dissociation of stalled RNAP from positively supercoiled DNA but not negatively supercoiled DNA. We observe that RapA-dependent RNAP dissociation is torque-sensitive, is inhibited by GreB and depends on RNA length. We propose that stalled RNAP is dislodged from DNA by RapA via backtracking in a supercoiling- and torque-dependent manner, suggesting that RapA's activity on transcribing RNAP in vivo is responsible for resolving conflicts between converging polymerase molecular motors. |
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MeSH term(s) | DNA, Superhelical/metabolism ; DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/metabolism ; RNA/genetics ; RNA/metabolism ; Transcription, Genetic |
Chemical Substances | DNA, Superhelical ; Escherichia coli Proteins ; RapA protein, E coli ; RNA (63231-63-0) ; DNA-Directed RNA Polymerases (EC 2.7.7.6) |
Language | English |
Publishing date | 2022-07-12 |
Publishing country | England |
Document type | Journal Article ; Research Support, Non-U.S. Gov't |
ZDB-ID | 186809-3 |
ISSN | 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048 |
ISSN (online) | 1362-4962 ; 1362-4954 |
ISSN | 0301-5610 ; 0305-1048 |
DOI | 10.1093/nar/gkac558 |
Database | MEDical Literature Analysis and Retrieval System OnLINE |
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