mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Abstract
Data availability
Article and author information
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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.

The following data sets were generated

1. Loerch S
2. Bao C
3. Ling C
4. Korostelev AA
5. Grigorieff N
6. Ermolenko DN
(2020) 70S ribosome bound to HIV frameshifting stem-loop (FSS) and P-site tRNA (nonrotated conformation, Structure I)
Protein Data Bank, 6VWM.

http://www.rcsb.org/structure/6VWM
1. Loerch S
2. Bao C
3. Ling C
4. Korostelev AA
5. Grigorieff N
6. Ermolenko DN
(2020) 70S ribosome bound to HIV frameshifting stem-loop (FSS) and P-site tRNA (nonrotated conformation, Structure I)
EMDataBank, EMD-21421.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-21421
1. Loerch S
2. Bao C
3. Ling C
4. Korostelev AA
5. Grigorieff N
6. Ermolenko DN
(2020) 70S ribosome bound to HIV frameshifting stem-loop (FSS) and P-site tRNA (nonrotated conformation, Structure II)
Protein Data Bank, 6VWN.

http://www.rcsb.org/structure/6VWN
1. Loerch S
2. Bao C
3. Ling C
4. Korostelev AA
5. Grigorieff N
6. Ermolenko DN
(2020) 70S ribosome bound to HIV frameshifting stem-loop (FSS) and P-site tRNA (nonrotated conformation, Structure II)
EMDataBank, EMD-21422.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-21422
1. Loerch S
2. Bao C
3. Ling C
4. Korostelev AA
5. Grigorieff N
6. Ermolenko DN
(2020) 70S ribosome bound to HIV frameshifting stem-loop (FSS) and P/E tRNA (rotated conformation)
Protein Data Bank, 6VWL.

http://www.rcsb.org/structure/6VWL
1. Loerch S
2. Bao C
3. Ling C
4. Korostelev AA
5. Grigorieff N
6. Ermolenko DN
(2020) 70S ribosome bound to HIV frameshifting stem-loop (FSS) and P/E tRNA (rotated conformation)
EMDataBank, EMD-21420.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-21420

Article and author information

Author details

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.

"This ORCID iD identifies the author of this article:" 0000-0002-9224-8083
Sarah Loerch

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-1731-516X
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.
Andrei A Korostelev

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-1588-717X
Nikolaus Grigorieff

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

For correspondence
niko@grigorieff.org

Competing interests
Nikolaus Grigorieff, Reviewing editor, eLife.

"This ORCID iD identifies the author of this article:" 0000-0002-1506-909X
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.

"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.

Copyright

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.