1. Chromosomes and Gene Expression
  2. Structural Biology and Molecular Biophysics

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

  1. Inigo Urrutia-Irazabal
  2. James R Ault
  3. Frank Sobott
  4. Nigel J Savery
  5. Mark Simon Dillingham  Is a corresponding author
  1. University of Bristol, United Kingdom
  2. University of Leeds, United Kingdom
https://doi.org/10.7554/eLife.68829
Share this article
Cite this article
  1. Inigo Urrutia-Irazabal
  2. James R Ault
  3. Frank Sobott
  4. Nigel J Savery
  5. Mark Simon Dillingham
(2021)
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
eLife 10:e68829.
https://doi.org/10.7554/eLife.68829
  2. Download BibTeX
  3. Download .RIS

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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. The HDX-MS mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD025332.

The following data sets were generated

Article and author information

Author details

  1. Inigo Urrutia-Irazabal

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3653-1308

  2. James R Ault

    Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5131-438X

  3. Frank Sobott

    Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  4. Nigel J Savery

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0803-4075

  5. Mark Simon Dillingham

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    For correspondence
    mark.dillingham@bristol.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4612-7141

Funding

H2020 European Research Council (DNAREPAIRMAN)

  • Inigo Urrutia-Irazabal
  • Nigel J Savery
  • Mark Simon Dillingham

Biotechnology and Biological Sciences Research Council (BB/M012573/1)

  • James R Ault
  • Frank Sobott

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Urrutia-Irazabal et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,227
    views
  • 179
    downloads
  • 21
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Inigo Urrutia-Irazabal
  2. James R Ault
  3. Frank Sobott
  4. Nigel J Savery
  5. Mark Simon Dillingham
(2021)
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
eLife 10:e68829.
https://doi.org/10.7554/eLife.68829

Share this article

https://doi.org/10.7554/eLife.68829

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression

    Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome

    Kira A Cozzolino, Lynn Sanford ... Dylan J Taatjes
    Research Article

    Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by Trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS over a 75 min to 24 hr timeframe. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A (CA), via rapid suppression of IFN-responsive transcription factor (TF) activity. We also discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. To further probe Mediator kinase function, we completed cytokine screens and metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways in cell type-specific ways, and broad upregulation of anti-inflammatory lipid mediators occurred specifically in kinase-inhibited cells during hyperactive IFNγ signaling. A subset of these lipids (e.g. oleamide, desmosterol) serve as ligands for nuclear receptors PPAR and LXR, and activation of these receptors occurred specifically during hyperactive IFN signaling in CA-treated cells, revealing mechanistic links between Mediator kinases, lipid metabolism, and nuclear receptor function. Collectively, our results establish CDK8/CDK19 as context-specific metabolic regulators, and reveal that these kinases control gene expression not only via TFs, but also through metabolic changes and splicing. Moreover, we establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

    1. Chromosomes and Gene Expression

    Cryogenic Electron Microscopy: MagIC beads for scarce macromolecules

    Carlos Moreno-Yruela, Beat Fierz
    Insight

    Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.

    1. Chromosomes and Gene Expression

    Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

    Carmina Lichauco, Eric J Foss ... Antonio Bedalov
    Research Article

    The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.