Structures of translationally inactive mammalian ribosomes

  1. Alan Brown  Is a corresponding author
  2. Matthew R Baird
  3. Matthew CJ Yip
  4. Jason Murray
  5. Sichen Shao  Is a corresponding author
  1. Harvard Medical School, United States
  2. MRC Laboratory of Molecular Biology, United Kingdom

Abstract

The cellular levels and activities of ribosomes directly regulate gene expression during numerous physiological processes. The mechanisms that globally repress translation are incompletely understood. Here, we use electron cryomicroscopy to analyze inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation. We identify two types of ribosomes that are translationally repressed by protein interactions. The first comprises ribosomes sequestered with elongation factor 2 (eEF2) by SERPINE mRNA binding protein 1 (SERBP1) occupying the ribosomal mRNA entrance channel. The second type are translationally repressed by a novel ribosome-binding protein, interferon-related developmental regulator 2 (IFRD2), which spans the P and E sites and inserts a C-terminal helix into the mRNA exit channel to preclude translation. IFRD2 binds ribosomes with a tRNA occupying a noncanonical binding site, the 'Z site', on the ribosome. These structures provide functional insights into how ribosomal interactions may suppress translation to regulate gene expression.

Data availability

All cryo-EM maps and models have been deposited in EMDB under accession codes 9234, 9235, 9236, 9237, 9239, 9240, 9241 and 9242. All models have been deposited in PDB under accession codes 6MTB, 6MTC, 6MTD and 6MTE.

The following data sets were generated

Article and author information

Author details

  1. Alan Brown

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    alan_brown@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Matthew R Baird

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Matthew CJ Yip

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jason Murray

    Structural Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Sichen Shao

    Department of Cell Biology, Harvard Medical School, Boston, United States
    For correspondence
    sichen_shao@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2679-5537

Funding

Harvard Medical School (N/A)

  • Alan Brown
  • Matthew R Baird
  • Matthew CJ Yip
  • Sichen Shao

International Retinal Research Foundation (N/A)

  • Alan Brown

E. Matilda Ziegler Foundation for the Blind (N/A)

  • Alan Brown

Charles H. Hood Foundation (N/A)

  • Sichen Shao

Richard and Susan Smith Family Foundation (N/A)

  • Sichen Shao

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

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Version history

  1. Received: July 26, 2018
  2. Accepted: October 12, 2018
  3. Accepted Manuscript published: October 24, 2018 (version 1)
  4. Version of Record published: November 9, 2018 (version 2)

Copyright

© 2018, Brown 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

  • 6,991
    Page views
  • 992
    Downloads
  • 51
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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. Alan Brown
  2. Matthew R Baird
  3. Matthew CJ Yip
  4. Jason Murray
  5. Sichen Shao
(2018)
Structures of translationally inactive mammalian ribosomes
eLife 7:e40486.
https://doi.org/10.7554/eLife.40486

Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Elliott L Paine, Jack J Skalicky ... Wesley I Sundquist
    Research Advance

    The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease CAPN7 (Calpain-7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem MIT domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for: 1) CAPN7 recruitment to midbodies, 2) efficient abscission, and 3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Nina Gubensäk, Theo Sagmeister ... Tea Pavkov-Keller
    Research Article

    The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies e.g. bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.