1. Chromosomes and Gene Expression
  2. Structural Biology and Molecular Biophysics
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mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

  1. Chen Bao
  2. Sarah Loerch
  3. Clarence Ling
  4. Andrei A Korostelev
  5. Nikolaus Grigorieff  Is a corresponding author
  6. Dmitri N Ermolenko  Is a corresponding author
  1. University of Rochester, United States
  2. Janelia Research Campus, Howard Hughes Medical Institute, United States
  3. University of Massachusetts Medical School, United States
Research Article
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Cite this article as: eLife 2020;9:e55799 doi: 10.7554/eLife.55799

Abstract

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

Article and author information

Author details

  1. Chen Bao

    Department of Biochemistry and Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9224-8083
  2. Sarah Loerch

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1731-516X
  3. Clarence Ling

    Department of Biochemistry and Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, United States
    Competing interests
    No competing interests declared.
  4. Andrei A Korostelev

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1588-717X
  5. Nikolaus Grigorieff

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    For correspondence
    niko@grigorieff.org
    Competing interests
    Nikolaus Grigorieff, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X
  6. Dmitri N Ermolenko

    Department of Biochemistry and Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, United States
    For correspondence
    Dmitri_Ermolenko@urmc.rochester.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7554-5967

Funding

National Institute of General Medical Sciences (R01GM099719)

  • Dmitri N Ermolenko

National Institute of General Medical Sciences (5R35GM127094)

  • Andrei A Korostelev

Howard Hughes Medical Institute

  • Nikolaus Grigorieff

National Institute of Allergy and Infectious Diseases (P30 AI078498)

  • Dmitri N Ermolenko

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

Reviewing Editor

  1. Alan G Hinnebusch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States

Publication history

  1. Received: February 6, 2020
  2. Accepted: May 18, 2020
  3. Accepted Manuscript published: May 19, 2020 (version 1)

Copyright

© 2020, Bao 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.

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