Bacterial transcription is controlled by sigma factors, the RNA polymerase subunits that act as initiation factors. Although a single housekeeping sigma factor enables transcription from thousands of promoters, environmentally induced sigma factors redirect gene expression toward small regulons to carry out focused responses. Using structural and functional analyses, we determined the molecular basis of −10 promoter element recognition by Escherichia coli σE, which revealed an unprecedented way to achieve promoter melting. Group IV sigma factors induced strand separation at the −10 element by flipping out a single nucleotide from the nontemplate-strand DNA base stack. Unambiguous selection of this critical base was driven by a dynamic protein loop, which can be substituted to modify specificity of promoter recognition. This mechanism of promoter melting explains the increased promoter-selection stringency of environmentally induced sigma factors.
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We thank G. Schertler for his contribution in facilitating the collaboration between Eidgenössische Technische Hochschule (ETH) Zurich and the Paul Scherrer Institute (PSI), H.M. Fischer for helpful discussions, and V. Olieric for X-ray diffraction data collection for useful discussions and for reading the manuscript. This work was supported by the European Molecular Biology Organization postdoctoral fellowship ALTF 166–2012 (to S.C.), the Swiss National Science Foundation (SNF) through research grant 31003A–135623 (to J.A.V.) and the ETH research grant ETH–21 09–3 (to F.H.-T.A. and J.A.V.). The research project leading to the EPPIC server was supported by SNF grant 31003A_140879 and by PSI Research Committee grants FK05.08.1 and FK–04.09 to G.C.
The authors declare no competing financial interests.
Integrated supplementary information
(a–c) Sequences of dsDNA –10 promoter elements used for titration by σE2. The –10 promoter element is shown in (a), the C–10T and the A–112–AP variants in (b) and (c), respectively. 2–AP stands for 2–Amino Purine. (d–f) Overlays of the 1D 1H spectra that show imino proton signals of the DNA before (black) and after addition of one equimolar amount of σE2 (red). Spectra corresponding to the –10 promoter element, the C–10T and the A–112–AP variants are shown in (d), (e) and (f) respectively. (g–i) Overlay of the 2D 1H–1H TOCSY spectra showing the chemical shifts of H5–H6 protons of cytosine before (black) and after addition of one equimolar amount of σE2 (red). Spectra corresponding to the –10 promoter element, the C–10T and the A–112–AP variants are shown in (g), (h) and (i), respectively. (j–l) Overlay of the portion of the 2D 15N–1H HSQC spectra that shows the signal of the W73 side chain before (black) and after addition of one equimolar amount of σE2 (red). Spectra corresponding to titrations realized with the –10 promoter element, the C–10T and the A–112–AP variants are shown in (j), (k) and (l), respectively. (m–o) 1D 1H spectra showing the signal of the methyl group of I77 after addition of one equivalent amount of dsDNA. Spectra corresponding to titration realized with the –10 promoter element, the C–10T and the A–112–AP variants are shown in (m), (n) and (o), respectively.
Supplementary Figure 2 High specificity of the interaction between σE2 and its cognate –10 promoter element NT strand.
(a-g) ITC titrations of σE2 by different versions of the –10 promoter element nt strand. σE2 was titrated with the wild type –10 promoter element nt strand (5′–T–13G–12T–11C–10A–9A–8A–7–3′) in a. For other titrations, the nucleotide substitution is indicated on the panel of each titration. (h) Table summarizing values of dissociation constants. The affinity factor corresponds to the ratio between the KD (dissociation constant) observed and the KD observed for the wild type –10 promoter element nt strand. (i) Comparison of the chemical shift perturbations (Csp) observed during NMR titration of σE2 by its cognate nt strand (black) or by the mutated version C–10T of the –10 promoter element nt strand (red). (j) ITC titration of σE2 by the dsDNA form of the –10 promoter element.
Supplementary Figure 3 Crystal, electron density maps and electrostatic potential of the σE2–TGTCAAA complex.
(a) Pictures of the crystals containing the complex formed by σE2 and its cognate –10 promoter element nt strand. The left panel shows crystals in the NMR tube and the right panel corresponds to a zoomed view of two selected crystals. (b) Ribbon representation of the structure of the protein–ssDNA complex. The electron density map 2mFo–DFc corresponding to the ssDNA part is shown in yellow and illustrated as stick models. The protein is shown in gray and water molecules as red spheres. (c) Stick model representation of the protein–ssDNA complex. In this panel, the electron density map 2mFo–DFc corresponding to the protein is illustrated. (d) Electrostatic surface potential of the protein in the protein–ssDNA complex. The surface of the protein is colored in function of the electrostatic potential and the ssDNA is represented as stick models in yellow.
(a) Autoradiography of the 6% acrylamide denaturing RNA gel showing the presence of a radiolabelled RNA (indicated by the black arrowhead) only when all the components of the reaction were added. (b) Autoradiography of the 6% acrylamide denaturing RNA gels showing results of in vitro transcription assays realized with σE and different promoter variants. Promoter substitutions are indicated at the top of the gel. (c) Autoradiography of the 6% acrylamide denaturing RNA gels showing results of in vitro transcription assays realized with different σE variants. Protein mutations are indicated at the top of the gel. (d) Autoradiography of the 6% acrylamide denaturing RNA gels showing results of in vitro transcription assays with σE and the different chimeric proteins. Position of the transcript is indicated by a black arrowhead.
(a) Overlay of the aliphatic part of the 2D 13C–1H HSQC recorded with the free protein (black spectrum) or in the context of the σE2–TGTCAAA complex (red spectra). Positions of the signals corresponding to the aliphatic side chain of I77 are indicated. (b) Overlay of the aromatic part of 2D 13C–1H HSQC recorded with the free σE2 (black spectrum) or in the context of the σE2–TGTCAAA complex (red spectra). Positions of the signals corresponding to the aromatic cycle of Y75 are indicated. (c) NMR titration of the region 2 of σEcfG by the –10 promoter element nt strand 5′–TGGTTT–3′; 2D 15N–1H HSQC spectra of the protein recorded after each addition of ssDNA are displayed. (d-e) Closed–up views of the squared areas found in c.
Supplementary Figure 6 Modeling of the dsDNA recognition of the upstream part of the –10 promoter element and structural comparison of the position of equivalent thymine T–11 and T–12.
(a) Model of dsDNA recognition of the upstream part of the –10 promoter element. The base pair –12 is displayed. (b) Superimposition of the helical hinge α3–loop L3–α4 of primary (gray) and ECF ss (gold) and of the two bases specifically recognized (T–11 and C–10 for σE and T–12 and A–11 for the primary s).
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Campagne, S., Marsh, M., Capitani, G. et al. Structural basis for −10 promoter element melting by environmentally induced sigma factors. Nat Struct Mol Biol 21, 269–276 (2014). https://doi.org/10.1038/nsmb.2777
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