1. Structural Biology and Molecular Biophysics
Download icon

Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch

  1. My-Tra Le  Is a corresponding author
  2. Wojciech K Kasprzak
  3. Taejin Kim
  4. Feng Gao
  5. Megan YL Young
  6. Xuefeng Yuan
  7. Bruce A Shapiro
  8. Joonil Seog
  9. Anne E Simon  Is a corresponding author
  1. University of Maryland, United States
  2. Leidos Biomedical Research, Inc., United States
  3. National Cancer Institute, United States
  4. College of Plant Protection, Shandong Agricultural University, China
Research Article
  • Cited 7
  • Views 860
  • Annotations
Cite this article as: eLife 2017;6:e22883 doi: 10.7554/eLife.22883

Abstract

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.

Article and author information

Author details

  1. My-Tra Le

    Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States
    For correspondence
    my.letra@gmail.com
    Competing interests
    The authors declare that no competing interests exist.
  2. Wojciech K Kasprzak

    Basic Science Program, Leidos Biomedical Research, Inc., Frederick, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Taejin Kim

    RNA Biology Laboratory, National Cancer Institute, Frederick, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Feng Gao

    Department of Cell Biology and Molecular Genetics, University of Maryland, College Pak, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Megan YL Young

    Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Xuefeng Yuan

    Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Tai'an, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Bruce A Shapiro

    RNA Biology Laboratory, National Cancer Institute, Frederick, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Joonil Seog

    Department of Materials Science and Engineering, University of Maryland, College Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Anne E Simon

    Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States
    For correspondence
    simona@umd.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6121-0704

Funding

National Science Foundation (MCB-1411836)

  • My-Tra Le
  • Feng Gao
  • Megan YL Young
  • Xuefeng Yuan
  • Anne E Simon

National Institutes of Health (R21AI117882-01)

  • My-Tra Le
  • Feng Gao
  • Anne E Simon

National Cancer Institute (Intramural)

  • Wojciech K Kasprzak
  • Taejin Kim
  • Bruce A Shapiro

National Institutes of Health (T32GM080201)

  • Megan YL Young

National Institutes of Health (2T32AI051967-06A1)

  • Megan YL Young
  • Anne E Simon

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

Publication history

  1. Received: November 3, 2016
  2. Accepted: February 7, 2017
  3. Accepted Manuscript published: February 10, 2017 (version 1)
  4. Accepted Manuscript updated: February 13, 2017 (version 2)
  5. Version of Record published: March 3, 2017 (version 3)

Copyright

© 2017, Le 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

  • 860
    Page views
  • 220
    Downloads
  • 7
    Citations

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

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)

  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Thuy-Lan V Lite et al.
    Research Article

    Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

    1. Microbiology and Infectious Disease
    2. Structural Biology and Molecular Biophysics
    William Wan et al.
    Research Article Updated

    Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles.