Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA

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 fo...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 118; no. 15; p. e2019630118
Main Authors Portman, James R, Brouwer, Gwendolyn M, Bollins, Jack, Savery, Nigel J, Strick, Terence R
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 13.04.2021
Subjects
Biological Sciences
Deoxyribonucleic acid
DNA
DNA-directed RNA polymerase
Domains
Hybrids
Mfd protein
Mutagenesis
Nucleic acids
Nucleotide sequence
Partitioning
Proteins
R-loops
Regulatory sequences
Ribonucleic acid
RNA
RNA polymerase
Supercoiling
Tethering
single molecule
magnetic trapping
R-loops
mutagenesis
transcription-coupled repair
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Summary: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.
Bibliography:Edited by Aziz Sancar, University of North Carolina at Chapel Hill, Chapel Hill, NC, and approved February 5, 2021 (received for review September 17, 2020)
Author contributions: J.R.P., N.J.S., and T.R.S. designed research; J.R.P., G.M.B., and J.B. performed research; J.R.P., G.M.B., J.B., N.J.S., and T.R.S. analyzed data; and J.R.P., N.J.S., and T.R.S. wrote the paper.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2019630118