A full-length group 1 bacterial sigma factor adopts a compact structure incompatible with DNA binding

Abstract

The sigma factors are the key regulators of bacterial transcription initiation. Through direct read-out of promoter DNA sequence, they recruit the core RNA polymerase to sites of initiation, thereby dictating the RNA polymerase promoter-specificity. The group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an N-terminal sequence known as sigma1.1. We report the solution structure of Thermotoga maritima sigmaA sigma1.1. We additionally demonstrate by using chemical crosslinking strategies that sigma1.1 is in close proximity to the promoter recognition domains of sigmaA. We therefore propose that sigma1.1 autoinhibits promoter DNA binding of free sigmaA by stabilizing a compact organization of the sigma factor domains that is unable to bind DNA.

Figure 1. The structure of σ_1.1

(A) An alignment of σ_1.1 from five different bacterial species generated using the ClustalW program. Highlighted in red are identical residues, in orange are conserved residues and in yellow are semi-conserved residues. The black line below the alignment represents the construct used for structural determination, with secondary structural elements (H1–H3) indicated. (B) A superimposition of the backbone of the 20 lowest energy structures of σ_1.1 obtained from calculations using NMR-derived restraints. Each structure is individually colored. The N-termini are in the upper left and the C-termini are in the bottom half of the figure. (C) Cartoon representation of the secondary structure elements of σ_1.1. The structure is rainbow spectrum colored from N-terminus (blue) to C-terminus (red). (D) Cartoon representation of σ_1.1. Residues that form the hydrophobic core are shown in yellow. Images generated using MacPYMOL (Delano Scientific). (E) The electrostatic surface of σ_1.1. Red is negative, blue is positive, white is neutral. Image generated using GRASP (Nicholls et al., 1991).

Figure 2. Crosslinking to detect intramolecular interactions

(A) Chemical structure of the photo-crosslinker used in this study. A synthetic scheme is provided in supplemental material. (B) The crosslinker (red star) is attached by disulfide exchange to a surface cysteine in the σ factor. Following photo-crosslinking and digestion, the disulfide is reduced, transferring the tag to regions in close proximity to σ_1.1. The transferred-tag is detected by electrophoretic methods. (C) σ_1.1 residues mutated for crosslinking studies. The surface of σ_1.1 is rendered in blue with residues that were mutated to cysteine for attachment of the crosslinker highlighted in red. Images were generated using MacPYMOL (Delano Scientific). (D) Crosslinking can be induced by uv irradiation. Indicated σ^A mutants were labeled with crosslinker by disulfide exchange. Samples that had either been or had not been irradiated at 325 nm light were then reduced with DTT. Samples were then resolved by SDS-PAGE and analyzed by Western blot (Streptavidin-HRP).

Figure 3. Interdomain crosslinking from σ_1.1

(A) Predicted CNBr digestion of Tm σ^A. The domain structure of σ^A (top) is shown with the predicted CNBr digestion fragments below. Residue numbers of cleavage products are shown with molecular weights in parenthesis. (B) Photo-crosslinking from σ_1.1 analyzed by label transfer and CNBr digestion. Crosslinker was attached at the indicated residues within σ_1.1 through a disulfide. Wild-type σ^A, which lacks a native cysteine, was included as a control. Irradiated (UV+) and non-irradiated (UV−) samples were digested with CNBr, separated by reducing SDS-PAGE (10–20% acrylamide, tris-tricine buffer system) and probed for biotin by Western blotting (HRP-streptavidin). Note, only the irradiated cysteine mutants retained the biotin. The identity of the labeled bands is shown at right. Bands that have been identified by mass spectrometry are labeled with residue numbers. A full account of the assignment protocol is given in supplemental methods.

Figure 4. BNPS-Skatole and two-stage digestion of crosslinked products

(A) BNPS-skatole cleaves after Trp residues and, as a consequence, conveniently cuts σ^A between σ₂ and σ₃. (B) Photo-crosslinking from σ_1.1 analyzed by label transfer and BNPS digestion. Crosslinker was attached at the indicated residues within σ_1.1 through a disulfide. Wild-type σ^A, which lacks a native cysteine, was included as a control. Irradiated (UV+) and non-irradiated (UV−) samples were digested with BNPS, separated by reducing SDS-PAGE (4–12% acrylamide) and probed for biotin by Western blotting (HRP-streptavidin). Note, the D60C sample did not label well, so a longer exposure is shown to the right. The identity of the labeled bands (shown at right) was determined by in-gel trypsinolysis followed by mass spectrometry (data not shown). (C,D) Two-step digestion protocol. Irradiated crosslinker samples of the indicated attachment sites were digested with BNPS-skatole, separated by SDS-PAGE (4–12% acrylamide). Following excision and extraction from the gel, the C-terminal BNPS-skatole fragment (panel C) and N-terminal BNPS-skatole fragment (panel D) were further digested with CNBr and analyzed by SDS-PAGE (10–20% acrylamide, tris-tricine buffer system) and Western blot (HRP-streptavidin).

Figure 5. Interdomain crosslinking from σ₄ and σ₂

Crosslinker was attached at the indicated residues within σ₄ (A) and σ₂ (B) through a disulfide. Irradiated (UV+) and non-irradiated (UV−) samples were digested with CNBr, separated by reducing SDS-PAGE (10–20% acrylamide, tris-tricine buffer system) and probed for biotin by Western blotting (HRP-streptavidin). The identity of the labeled bands is shown at right. The N[2–150]M, R[342–394]M and R[342–399]G bands were identified by mass spectrometry, the G[176–236]M band was identified as described in the text.

Figure 6. Proposed model of DNA binding inhibition by σ_1.1

(A) Crosslinker attachment sites are indicated on the electrostatic surface map of σ_1.1 generated using the GRASP program. Indicated in white are the sites that were shown to make interdomain crosslinks. Indicated in yellow are the sites from which no interdomain crosslinking was observed. (B) Schematic showing the compaction model of σ^A autoinhibition. The negative surface of σ_1.1 is capable of forming crosslinks to the DNA binding domains σ₂ and σ₄. It is thus likely that σ_1.1 organizes the σ factor into a compacted structure that is incapable of binding DNA. σ₂ forms crosslinks to σ₄, indicating that these two domains must also be in close proximity.