NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA

  1. Zachary F Mandell
  2. Reid T Oshiro
  3. Alexander V Yakhnin
  4. Rishi Vishwakarma
  5. Mikhail Kashlev
  6. Daniel B Kearns
  7. Paul Babitzke  Is a corresponding author
  1. The Pennsylvania State University, United States
  2. Indiana University, United States
  3. National Cancer Institute, United States

Abstract

NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, DnusG, and NusA depletion DnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.

Data availability

RNA-seq data were deposited in GEO under accession number GSE154522. All other data generated or analysed during this study are included in the manuscript and supporting files.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Zachary F Mandell

    Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Reid T Oshiro

    Department of Biology, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Alexander V Yakhnin

    NCI RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7313-7054
  4. Rishi Vishwakarma

    Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Mikhail Kashlev

    NCI RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Daniel B Kearns

    Department of Biology, Indiana University, Bloomington, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Paul Babitzke

    Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, United States
    For correspondence
    pxb28@psu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2481-1062

Funding

National Institutes of Health (GM098399)

  • Paul Babitzke

National Institutes of Health (GM131783)

  • Daniel B Kearns

National Institutes of Health (intramural)

  • Mikhail Kashlev

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

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 5,427
    views
  • 473
    downloads
  • 41
    citations

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

Download links

Share this article

https://doi.org/10.7554/eLife.61880

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Eva Herdering, Tristan Reif-Trauttmansdorff ... Ruth Anne Schmitz
    Research Article

    Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.