Abstract

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

Data availability

The cryo-EM density maps have been deposited in the EMDataBank under accession codes EMD-21996 [L1(ATP)], EMD-22006 [L2(ADP)], EMD-22012 [I(ATP)], EMD-22039 [II(ATP)], EMD-22043 [III(ADP)], EMD-22044 [IV(ADP)], and EMD-22045 [V(ATP)]. The atomic coordinates have been deposited in the Protein Data Bank under accession codes 6X26 [L1(ATP)], 6X2F [L2(ADP)], 6X2N [I(ATP)], 6X43 [II(ATP)], 6X4W [III(ADP)], 6XYY [IV(ADP)], and 6X5Q [V(ATP)].

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Jin Young Kang

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Eliza Llewellyn

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. James Chen

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Paul D B Olinares

    Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3429-6618
  5. Joshua Brewer

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Brian T Chait

    Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Elizabeth A Campbell

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Seth A Darst

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    For correspondence
    darst@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8241-3153

Funding

National Institute of General Medical Sciences (P41 GM109824)

  • Brian T Chait

National Institute of General Medical Sciences (P41 GM103314)

  • Brian T Chait

National Institute of General Medical Sciences (R01 GM114450)

  • Elizabeth A Campbell

National Institute of General Medical Sciences (R35 GM118130)

  • Seth A Darst

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

Reviewing Editor

  1. James M Berger, Johns Hopkins University School of Medicine, United States

Version history

  1. Received: August 14, 2020
  2. Accepted: January 21, 2021
  3. Accepted Manuscript published: January 22, 2021 (version 1)
  4. Version of Record published: February 4, 2021 (version 2)

Copyright

© 2021, Kang 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

  • 2,545
    views
  • 345
    downloads
  • 34
    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. Jin Young Kang
  2. Eliza Llewellyn
  3. James Chen
  4. Paul D B Olinares
  5. Joshua Brewer
  6. Brian T Chait
  7. Elizabeth A Campbell
  8. Seth A Darst
(2021)
Structural basis for transcription complex disruption by the Mfd translocase
eLife 10:e62117.
https://doi.org/10.7554/eLife.62117

Share this article

https://doi.org/10.7554/eLife.62117

Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Dimitrios Vismpas, Friedrich Förster
    Insight

    Advanced cryo-EM approaches reveal surprising insights into the molecular structure that allows nascent proteins to be inserted into the membrane of the endoplasmic reticulum.

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics
    Samuel C Griffiths, Jia Tan ... Hsin-Yi Henry Ho
    Research Article

    The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.