E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

  1. James Chen
  2. Saumya Gopalkrishnan
  3. Courtney Chiu
  4. Albert Y Chen
  5. Elizabeth A Campbell
  6. Richard L Gourse
  7. Wilma Ross
  8. Seth A Darst  Is a corresponding author
  1. The Rockefeller University, United States
  2. University of Wisconsin-Madison, United States

Abstract

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

Data availability

The cryo-EM density maps have been deposited in the EMDataBank under accession codes EMD-0348 [Eco TraR-Eσ70(I)], EMD-0349 [Eco TraR-Eσ70(II)], EMD-20231 [Eco TraR-Eσ70(III)], EMD-20230 (Eco Eσ70), EMD-20203 (rpsT P2-RPo), and EMD-20232 (rpsT P2-RPo2). The atomic coordinates have been deposited in the Protein Data Bank under accession codes 6N57 [Eco TraR-Eσ70(I)], 6N58 [Eco TraR-Eσ70(II)], 6P1K (Eco Eσ70), and 6OUL (rpsT P2-RPo).

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

Article and author information

Author details

  1. James Chen

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2311-003X
  2. Saumya Gopalkrishnan

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Courtney Chiu

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Albert Y Chen

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Elizabeth A Campbell

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Richard L Gourse

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Wilma Ross

    Department of Bacteriology, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Seth A Darst

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, United States
    For correspondence
    darst@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8241-3153

Funding

National Institutes of Health (R01 GM114450)

  • Elizabeth A Campbell

National Institutes of Health (R01 GM37048)

  • Richard L Gourse

National Institutes of Health (R35 GM118130)

  • Seth A Darst

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

Copyright

© 2019, Chen 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

  • 3,080
    views
  • 494
    downloads
  • 56
    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. James Chen
  2. Saumya Gopalkrishnan
  3. Courtney Chiu
  4. Albert Y Chen
  5. Elizabeth A Campbell
  6. Richard L Gourse
  7. Wilma Ross
  8. Seth A Darst
(2019)
E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation
eLife 8:e49375.
https://doi.org/10.7554/eLife.49375

Share this article

https://doi.org/10.7554/eLife.49375

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Santi Mestre-Fos, Lucas Ferguson ... Jamie HD Cate
    Research Article

    Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Ana Patrícia Graça, Vadim Nikitushkin ... Gerald Lackner
    Research Article

    Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.