Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops

Abstract

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the β subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimise conflicts between transcription and other processes on DNA including replication.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

“…subtilis RNaseJ1 clears stalled RNAP using a torpedo mechanism (5′–3′ exonuclease activity followed by RNAP displacement) (Sikova et al, 2020), and in Escherichia coli the helicase protein RapA is important in recycling RNAP (Liu et al, 2015). UvrD/PcrA in concert with Gre factors has been reported to act on RNAP stalled at a DNA lesion, binding to the complex and using the energy of ATP hydrolysis to backtrack away from the lesion to allow repair systems access to the damaged DNA (Epshtein et al, 2014; Hawkins et al, 2019), although it now appears that the role of these helicases is in preventing the formation of, and resolving, R‐loops (RNA‐DNA hybrids) that can have a detrimental effect on DNA replication (Urrutia‐Irazabal et al, 2021).…”

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

“…In B. subtilis RNaseJ1 clears stalled RNAP using a torpedo mechanism (5'-3' exonuclease activity followed by RNAP displacement) (Sikova et al, 2020), and in Escherichia coli the helicase protein RapA has been shown to be important in recycling RNAP (Liu et al, 2015). UvrD/PcrA in concert with Gre factors has been reported to act on RNAP stalled at a DNA lesion, binding to the complex and using the energy of ATP hydrolysis to backtrack away from the lesion to allow repair systems access to the damaged DNA (Epshtein et al, 2014, Hawkins et al, 2019, although it now appears that the role of these helicases is in preventing formation of, and resolving, R-loops (RNA-DNA hybrids) that can have a detrimental effect on DNA replication (Urrutia-Irazabal et al, 2021).…”