Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria

  1. Matthew V Kotlajich
  2. Daniel R Hron
  3. Beth A Boudreau
  4. Zhiqiang Sun
  5. Yuri Lyubchenko
  6. Robert Landick  Is a corresponding author
  1. University of Wisconsin-Madison, United States
  2. University of Nebraska Medical Center, United States

Abstract

Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. Here we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria.

Article and author information

Author details

  1. Matthew V Kotlajich

    Biochemistry, University of Wisconsin-Madison, Department of Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Daniel R Hron

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Beth A Boudreau

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Zhiqiang Sun

    Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Yuri Lyubchenko

    Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Robert Landick

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    For correspondence
    landick@biochem.wisc.edu
    Competing interests
    The authors declare that no competing interests exist.

Copyright

© 2015, Kotlajich 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

  • 3,279
    views
  • 582
    downloads
  • 119
    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. Matthew V Kotlajich
  2. Daniel R Hron
  3. Beth A Boudreau
  4. Zhiqiang Sun
  5. Yuri Lyubchenko
  6. Robert Landick
(2015)
Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria
eLife 4:e04970.
https://doi.org/10.7554/eLife.04970

Share this article

https://doi.org/10.7554/eLife.04970

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. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    A Sofia F Oliveira, Fiona L Kearns ... Adrian J Mulholland
    Short Report

    The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.