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

Abstract

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type σ subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains σ in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the σ subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

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) E_PR 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 E_PR and R are shown on the right.

Fig. 2. RbpA is required for “opening” of σ^B in the MtbRNAP holoenzyme.

(A) E_PR histograms for free σ^B, RbpA-σ^B complex, and MtbRNAP-σ^B and RbpA-MtbRNAP-σ^B 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 σ^B subunit. (C) E_PR histogram of chimeric EcoRNAP-σ^B holoenzyme.

Fig. 3. Interaction with promoter DNA stabilizes the open conformation of the σ^B 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 σ^B [wild-type (WT)] and donor-acceptor–labeled σ^B (Dy). (C) Quantification of the gel shown in (B). Values were normalized to that obtained for MtbRNAP-σ^B in the presence of RbpA. (D) E_PR histograms for MtbRNAP-σ^B in complex with us-fork without (+us-fork) or with RbpA (+us-fork + RbpA). (E) E_PR histograms for MtbRNAP-σ^B 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.