Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

  1. James R Fuller
  2. Phoebe A Rice  Is a corresponding author
  1. The University of Chicago, United States

Abstract

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

Article and author information

Author details

  1. James R Fuller

    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Phoebe A Rice

    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States
    For correspondence
    price@uchicago.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3467-341X

Funding

National Institute of General Medical Sciences (GM101989)

  • James R Fuller
  • Phoebe A Rice

National Center for Research Resources (1S10RR026988-01)

  • James R Fuller
  • Phoebe A Rice

National Science Foundation (DMR1420709)

  • James R Fuller
  • Phoebe A Rice

National Institute of General Medical Sciences (GM007183)

  • James R Fuller
  • Phoebe A Rice

National Institute of General Medical Sciences (GM104397)

  • James R Fuller
  • Phoebe A Rice

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

Reviewing Editor

  1. David Sherratt, University of Oxford, United Kingdom

Version history

  1. Received: September 23, 2016
  2. Accepted: February 7, 2017
  3. Accepted Manuscript published: February 8, 2017 (version 1)
  4. Accepted Manuscript updated: February 13, 2017 (version 2)
  5. Version of Record published: March 17, 2017 (version 3)

Copyright

© 2017, Fuller & Rice

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

  • 1,620
    Page views
  • 329
    Downloads
  • 19
    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)

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. James R Fuller
  2. Phoebe A Rice
(2017)
Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal
eLife 6:e21777.
https://doi.org/10.7554/eLife.21777

Share this article

https://doi.org/10.7554/eLife.21777

Further reading

    1. Biochemistry and Chemical Biology
    Jake W Anderson, David Vaisar ... Natalie G Ahn
    Research Article

    Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

    1. Biochemistry and Chemical Biology
    Anne E Hultgren, Nicole MF Patras, Jenna Hicks
    Feature Article

    Organizations that fund research are keen to ensure that their grant selection processes are fair and equitable for all applicants. In 2020, the Arnold and Mabel Beckman Foundation introduced blinding to the first stage of the process used to review applications for Beckman Young Investigator (BYI) awards: applicants were instructed to blind the technical proposal in their initial Letter of Intent by omitting their name, gender, gender-identifying pronouns, and institutional information. Here we examine the impact of this change by comparing the data on gender and institutional prestige of the applicants in the first four years of the new policy (BYI award years 2021–2024) with data on the last four years of the old policy (2017–2020). We find that under the new policy, the distribution of applicants invited to submit a full application shifted from those affiliated with institutions regarded as more prestigious to those outside of this group, and that this trend continued through to the final program awards. We did not find evidence of a shift in the distribution of applicants with respect to gender.