Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA

Proc Natl Acad Sci U S A. 2023 Jul 11;120(28):e2303849120. doi: 10.1073/pnas.2303849120. Epub 2023 Jul 5.

Abstract

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

Keywords: RapA; kinetic modeling; post termination complex; single-molecule spectroscopy; transcription.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • DNA / metabolism
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / genetics
  • Escherichia coli Proteins* / metabolism
  • Kinetics
  • RNA, Bacterial* / metabolism
  • Transcription, Genetic

Substances

  • RNA, Bacterial
  • DNA-Directed RNA Polymerases
  • Adenosine Triphosphatases
  • DNA
  • Adenosine Triphosphate
  • RapA protein, E coli
  • Escherichia coli Proteins