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

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.

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

  • 1,090
    views
  • 241
    downloads
  • 14
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. My-Tra Le
  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
(2017)
Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch
eLife 6:e22883.
https://doi.org/10.7554/eLife.22883

Share this article

https://doi.org/10.7554/eLife.22883

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

    1. Structural Biology and Molecular Biophysics
    Christopher T Schafer, Raymond F Pauszek III ... David P Millar
    Research Article

    The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G-protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.