Research ArticleSTRUCTURAL BIOLOGY

Single-molecule analysis reveals the mechanism of transcription activation in M. tuberculosis

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Science Advances  23 May 2018:
Vol. 4, no. 5, eaao5498
DOI: 10.1126/sciadv.aao5498
  • Fig. 1 The σ70 subunit in the RNAP holoenzyme exhibits conformational heterogeneity.

    (A) Scheme of the σ subunits. Positions of the Cys substitutions are underlined. (B) Structure of the EcoRNAP-σ70 holoenzyme [Protein Data Bank (PDB) code: 4IGC (35)] and (C) its complex with us-fork. The subunits of the RNAP core are shown as molecular surfaces, and the σ subunit is shown as ribbons in cyan. The Cα atoms of the σ subunit residues labeled by fluorophores are shown as spheres in green (σ domain 4) and magenta (σ domain 2). The distance between the Cα atoms is indicated. (D) Sequence of the us-fork DNA. The −10 and −35 promoter elements are underlined and shaded. (E) EPR histograms for free σ70, EcoRNAP-σ70 holoenzyme, and its complex with us-fork (+us-fork). Two conformations of σ, corresponding to the open and closed states, are shown schematically on the top. The black thick lines show Gaussian fits of smFRET efficiencies for individual subpopulations, and the dashed lines represent the sum of Gaussians for the overall population. Mean peak EPR and R are shown on the right.

  • Fig. 2 RbpA is required for “opening” of σB in the MtbRNAP holoenzyme.

    (A) EPR histograms for free σB, RbpA-σB complex, and MtbRNAP-σB and RbpA-MtbRNAP-σB complex. (B) Structure of Mycobacterium smegmatis RNAP in complex with RbpA [PDB code: 5TW1 (22)]. Labeling as in Fig. 1B. Residue numbering corresponds to the σB subunit. (C) EPR histogram of chimeric EcoRNAP-σB holoenzyme.

  • Fig. 3 Interaction with promoter DNA stabilizes the open conformation of the σB subunit.

    (A) Structure of M. smegmatis RNAP in complex with RbpA and us-fork (blue, template strand; red, nontemplate strand) (PDB code: 5TW1). (B) Native gel electrophoresis analysis of the promoter complex formation between labeled (+Cy3) and unlabeled (−Cy3) us-fork DNA and RNAP holoenzymes containing σB [wild-type (WT)] and donor-acceptor–labeled σB (Dy). (C) Quantification of the gel shown in (B). Values were normalized to that obtained for MtbRNAP-σB in the presence of RbpA. (D) EPR histograms for MtbRNAP-σB in complex with us-fork without (+us-fork) or with RbpA (+us-fork + RbpA). (E) EPR histograms for MtbRNAP-σB in complex with sigAP promoter without (+sigAP) or with RbpA (+RbpA).

  • Fig. 4 Effect of promoter architecture on MtbRNAP activity.

    (A) Native gel electrophoresis analysis of the MtbRNAP complex formation with Cy3-labeled us-fork and us-fork harboring extended −10 element (Ext −10). RbpA was added either before (R + F) or after (F + R) the us-fork DNA. (B) Quantification of the gel shown in (A). Values were normalized to that obtained in the presence of RbpA for each template separately. (C) Run-off [32P]RNA products synthesized in multiple-round transcription from the wild-type sigAP promoter and its derivative harboring extended −10 element. (D) Quantification of the RNA products shown in (C). Values were normalized to that obtained for the wild-type sigAP promoter in the presence of RbpA. RbpA was added either before (R + P) or after (P + R) the promoter DNA.

  • Fig. 5 The “dynamic” model for the σ subunit activation and promoter recognition.

    (A) Activator-independent mechanism used by E. coli RNAP. (B) Activator-dependent and activator-independent mechanisms used by MtbRNAP.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaao5498/DC1

    fig. S1. Incorporation of the fluorescent dyes into σ70 and σB subunits.

    fig. S2. Activity of the σ70 and σB double-Cys mutants and their labeled derivatives.

    fig. S3. Test of protein oligomerization using MB analysis on the labeled species.

    fig. S4. Calculation of the donor-acceptor distances using FPS software.

    fig. S5. Analysis of the RNAP holoenzyme assembly by the ssFCS.

    fig. S6. Interaction of RNAP with an us-fork template.

    table S1. Apparent distances (Rap) between domains σ2 and σ4 determined in ensemble LRET experiments.

    table S2. Summary of the donor-acceptor distances for σ70 C442-C579.

    table S3. Summary of the donor-acceptor distances for σB C151-C292.

    Reference (40)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Incorporation of the fluorescent dyes into σ70 and σB subunits.
    • fig. S2. Activity of the σ70 and σB double-Cys mutants and their
      labeled derivatives.
    • fig. S3. Test of protein oligomerization using MB analysis on the labeled species.
    • fig. S4. Calculation of the donor-acceptor distances using FPS software.
    • fig. S5. Analysis of the RNAP holoenzyme assembly by the ssFCS.
    • fig. S6. Interaction of RNAP with an us-fork template.
    • table S1. Apparent distances (Rap) between domains σ2 and σ4 determined in ensemble LRET experiments.
    • table S2. Summary of the donor-acceptor distances for σ70 C442-C579.
    • table S3. Summary of the donor-acceptor distances for σB C151-C292.
    • Reference (40)

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