mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding
- University of Rochester, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- University of Massachusetts Medical School, United States
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.
Data availability
Structural models have been deposited in PDB under the accession codes 6VWM, 6VWN, 6VWL. Cryo-EM data have been deposited to EMDB under the accession codes EMD-21421, EMD-21422, EMD-21420.
Article and author information
Author details
Andrei A Korostelev
Nikolaus Grigorieff
Dmitri N Ermolenko
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
- Alan G Hinnebusch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
Version history
- Received: February 6, 2020
- Accepted: May 18, 2020
- Accepted Manuscript published: May 19, 2020 (version 1)
- Version of Record published: June 9, 2020 (version 2)
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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mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding
eLife 9:e55799.
https://doi.org/10.7554/eLife.55799
Further reading
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Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.
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