Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

  1. Fuzhou Ye
  2. Ioly Kotta-Loizou
  3. Milija Jovanovic
  4. Xiaojiao Liu
  5. David TF Dryden
  6. Martin Buck
  7. Xiaodong Zhang  Is a corresponding author
  1. Imperial College London, United Kingdom
  2. University of Durham, United Kingdom

Abstract

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.

Data availability

All data generated or analysed during the study are included in the manuscript and supporting files. The cryo EM maps and structural models will be deposited into EMDB and PDB with access codes 6R9G and 6R9B.

The following data sets were generated

Article and author information

Author details

  1. Fuzhou Ye

    Section of Structural Biology, Department of Medicine, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Ioly Kotta-Loizou

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Milija Jovanovic

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Xiaojiao Liu

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. David TF Dryden

    Department of Biosciences, University of Durham, Durham, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Martin Buck

    Life Sciences, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Xiaodong Zhang

    Section of Structural Biology, Department of Medicine, Imperial College London, London, United Kingdom
    For correspondence
    xiaodong.zhang@imperial.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9786-7038

Funding

Biotechnology and Biological Sciences Research Council (BB/N007816/1)

  • Fuzhou Ye

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

Copyright

© 2020, Ye 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

  • 2,450
    views
  • 343
    downloads
  • 15
    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. Fuzhou Ye
  2. Ioly Kotta-Loizou
  3. Milija Jovanovic
  4. Xiaojiao Liu
  5. David TF Dryden
  6. Martin Buck
  7. Xiaodong Zhang
(2020)
Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7
eLife 9:e52125.
https://doi.org/10.7554/eLife.52125

Share this article

https://doi.org/10.7554/eLife.52125

Further reading

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics
    Liza Dahal, Thomas GW Graham ... Xavier Darzacq
    Research Article

    Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Angel D'Oliviera, Xuhang Dai ... Jeffrey S Mugridge
    Research Article

    The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.