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1.
Nucleic Acids Res ; 50(13): 7396-7405, 2022 07 22.
Article in English | MEDLINE | ID: mdl-35819188

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.


Subject(s)
DNA, Superhelical , Escherichia coli Proteins/metabolism , Escherichia coli , DNA, Superhelical/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , RNA/genetics , RNA/metabolism , Transcription, Genetic
2.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Article in English | MEDLINE | ID: mdl-33827922

ABSTRACT

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.


Subject(s)
Bacterial Proteins/metabolism , R-Loop Structures , Transcription Factors/metabolism , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli , Mutation , Single Molecule Imaging , Transcription Factors/genetics , Transcription, Genetic
3.
J Mol Biol ; 430(22): 4496-4512, 2018 10 26.
Article in English | MEDLINE | ID: mdl-29733857

ABSTRACT

All active living organisms mitigate DNA damage via DNA repair, and the so-called nucleotide excision repair pathway represents a functionally major part of the cell's DNA repair repertoire [1]. In this pathway, the damaged strand of DNA is incised and removed before being resynthesized. This form of DNA repair requires a multitude of proteins working in a complex choreography. Repair thus typically involves detection of a DNA lesion, validation of that detection event, search for an appropriate incision site and subsequent DNA incision, DNA unwinding/removal, and DNA resynthesis and religation. These activities are ultimately the result of molecules randomly diffusing and bumping into each other and acting in succession. It is also true, however, that repair components are often assembled into functional complexes which may be more efficient or regular in their mode of action. Studying DNA repair complexes for their mechanisms of assembly, action, and disassembly can help address fundamental questions such as whether DNA repair pathways are branched or linear; whether, for instance, they tolerate fluctuations in numbers of components; and more broadly how search processes between macromolecules take place or can be enhanced.


Subject(s)
DNA Repair , Multiprotein Complexes/metabolism , Transcription, Genetic , Animals , DNA Damage , Humans , Models, Molecular , Protein Transport , Single Molecule Imaging
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