1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Mechanism of ribosome rescue by ArfA and RF2

  1. Gabriel Demo
  2. Egor Svidritskiy
  3. Rohini Madireddy
  4. Ruben Diaz-Avalos
  5. Timothy Grant
  6. Nikolaus Grigorieff
  7. Duncan Sousa
  8. Andrei A Korostelev  Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. Medicago, United States
  3. Janelia Research Campus, Howard Hughes Medical Institute, United States
  4. Florida State University, United States
Research Article
  • Cited 23
  • Views 2,193
  • Annotations
Cite this article as: eLife 2017;6:e23687 doi: 10.7554/eLife.23687
Voice your concerns about research culture and research communication: Have your say in our 7th annual survey.

Abstract

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA “senses” the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Gabriel Demo

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  2. Egor Svidritskiy

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  3. Rohini Madireddy

    Medicago, Durham, United States
    Competing interests
    No competing interests declared.
  4. Ruben Diaz-Avalos

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.
  5. Timothy Grant

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.
  6. Nikolaus Grigorieff

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    Nikolaus Grigorieff, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X
  7. Duncan Sousa

    Department of Biological Science, Florida State University, Tallahassee, United States
    Competing interests
    No competing interests declared.
  8. Andrei A Korostelev

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    andrei.korostelev@umassmed.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1588-717X

Funding

National Institutes of Health (GM106105)

  • Andrei A Korostelev

National Institutes of Health (GM107465)

  • Andrei A Korostelev

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

Reviewing Editor

  1. Rachel Green, Johns Hopkins School of Medicine, United States

Publication history

  1. Received: November 27, 2016
  2. Accepted: March 14, 2017
  3. Accepted Manuscript published: March 16, 2017 (version 1)
  4. Version of Record published: April 3, 2017 (version 2)
  5. Version of Record updated: April 4, 2017 (version 3)

Copyright

© 2017, Demo 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

  • 2,193
    Page views
  • 407
    Downloads
  • 23
    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)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    Hongki Song et al.
    Research Article

    Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/aSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Wan Hua Li et al.
    Research Article

    SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.