Abstract
A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1,2,3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12–14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4,5,6. Here we present cryo-electron microscopy structures of bacterial RNAP–promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2—universal structural features of RNAP—in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.
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Data availability
The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-9041 (Cor–RP2), EMD-9037 (RPo), EMD-9039 (RP2) and EMD-9047 (Cor-holo). The atomic coordinates have been deposited in the Protein Data Bank under accession codes 6EEC (Cor–RP2), 6EDT (RPo), 6EE8 (RP2) and 6M7J (Cor-holo).
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Acknowledgements
We thank M. Ebrahim and J. Sotiris at The Rockefeller University Evelyn Gruss Lipper Cryo-electron Microscopy Resource Center and E. Eng at the New York Structural Biology Center for help with cryo-EM data collection; A. Feklistov, M. Lilic, R. Saecker and other members of the Darst–Campbell laboratory, and R. Landick for helpful discussions of the manuscript. Some of the work reported here was conducted at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the NIH National Institute of General Medical Sciences (GM103310), NYSTAR, and the Simons Foundation (349247). This work was supported by NIH grants R35 GM118130 to S.A.D. and R01 GM114450 to E.A.C.
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H.B. expressed and purified proteins, performed biochemical assays, and prepared cryo-EM grids. H.B. and J.C. collected and processed cryo-EM data. R.J. prepared and validated Cor. H.B., S.A.D. and E.A.C. built, refined and validated the structures. S.A.D. and E.A.C. conceived the project. H.B., J.C., S.A.D. and E.A.C. prepared and revised the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Cor inhibits M. tuberculosis RNAP transcription initiation but not promoter DNA binding and data-processing pipeline for the cryo-EM movies of RNAP–σA–CarD–RbpA–Cor–AP3 promoter.
a, Chemical structure of Cor40. b, Abortive transcription initiation assays measuring GpUpU production in the presence of increasing concentrations of Cor. 32P-labelled abortive transcript production was monitored by polyacrylamide gel electrophoresis and autoradiography. Full gel is shown in Supplementary Fig. 1. c, Flow chart showing the image-processing pipeline for the cryo-EM data of M. tuberculosis RNAP–σA–CarD–RbpA–Cor–AP3 promoter complexes, starting with 3,718 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). Movies were frame-aligned and summed using MotionCor229. CTF estimation for each micrograph was calculated with Gctf30. A representative micrograph is shown following processing by MotionCor2. Particles were autopicked from each micrograph with Gautomatch and then sorted by 2D classification using RELION33 to assess quality. The twelve highest populated classes from the 2D classification are shown. After picking, the dataset contained 1,026,386 particles. A subset of particles was used to generate an ab initio map in cryoSPARC31. Using the low-pass-filtered (30 Å) ab initio map as a template, three rounds of 3D heterogeneous refinement were performed using cryoSPARC in a binomial-like fashion. One major, high-resolution class emerged, which was refined using cryoSPARC homogenous refinement and then sharpened for model building.
Extended Data Fig. 2 Cryo-EM of Cor–RP2.
a, Angular distribution calculated in cryoSPARC for particle projections. Heat map shows number of particles for each viewing angle. b, Gold-standard FSC41, calculated by comparing the two independently determined half maps from cryoSPARC. The dotted line represents the 0.143 FSC cut-off which indicates a nominal resolution of 3.6 Å. c, FSC calculated between the refined structure and the half map used for refinement (work, red), the other half map (free, blue) and the full map (black). d, Top left, the 3.6 Å-resolution cryo-EM density map of Cor–RP2. Top right, a cross-section of the structure, showing the DNA inside the RNAP cleft. Bottom, same views as above, but coloured by local resolution35. The boxed region in the right view is magnified on the far right and sliced at the level of the Cor-binding pocket. Density for the Cor molecule is outlined in red. e, Left, overview of the Cor–RP2 structure, shown as a molecular surface. The boxed region is magnified on the right. Right, magnified view of the Cor-binding pocket in the same orientation as the boxed region on the left. Proteins are shown as α-carbon backbone worms. Residues that interact with Cor are shown in stick format. Cor is shown in stick format with green carbon atoms. Hydrogen bonds are indicated by dashed grey lines. The cryo-EM difference density for the Cor is shown as green mesh.
Extended Data Fig. 3 Data-processing pipeline for the cryo-EM movies of RNAP–σA–CarD–RbpA–AP3 promoter (RPo and RP2) and cryo-EM of RPo.
a, Flow chart showing the image-processing pipeline for the cryo-EM data of RNAP–σA–CarD–RbpA–AP3 promoter complexes starting with 8,577 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). Movies were frame-aligned and summed using MotionCor229. CTF estimation for each micrograph was calculated with Gctf30. A representative micrograph is shown following processing by MotionCor2. Particles were autopicked from each micrograph with Gautomatch and then sorted by 2D classification using RELION33 to assess quality. The twelve highest-populated classes from the 2D classification are shown. The dataset contained 931,461 particles. A subset of particles was used to generate an ab initio map in cryoSPARC31. Using the low-pass-filtered (30 Å) ab initio map as a template, two rounds of 3D heterogeneous refinement were performed using cryoSPARC in a binomial-like fashion. Two major classes emerged, which were refined using cryoSPARC homogenous refinement and then sharpened for model building. b, Angular distribution calculated in cryoSPARC for particle projections. Heat map shows number of particles for each viewing angle. c, Gold-standard FSC41, calculated by comparing the two independently determined half maps from cryoSPARC. The dotted line represents the 0.143 FSC cut-off, which indicates a nominal resolution of 3.6 Å. d, FSC calculated between the refined structure and the half map used for refinement (work, red), the other half map (free, blue), and the full map (black). e, Top left, the 3.6 Å-resolution cryo-EM density map of RPo. Top right, cross-section of the structure, showing the DNA inside the RNAP cleft. Bottom, same views as above, but coloured by local resolution35.
Extended Data Fig. 4 Overall DNA path of RPo matches previously determined RPo structures.
Selected RPo structures containing a completely intact transcription bubble42,43 were superimposed with the cryo-EM RPo structure according to α-carbons of the structural core module (Extended Data Table 2). The resulting superposition of the nucleic acids is shown. The nucleic acids are shown as phosphate backbone worms, colour-coded as shown in the key.
Extended Data Fig. 5 Cryo-EM of RP2 class.
a, Angular distribution calculated in cryoSPARC for particle projections. Heat map shows number of particles for each viewing angle. b, Gold-standard FSC41, calculated by comparing the two independently determined half maps from cryoSPARC. The dotted line represents the 0.143 FSC cut-off, which indicates a nominal resolution of 3.9 Å. c, FSC calculated between the refined structure and the half map used for refinement (work, red), the other half map (free, blue), and the full map (black). d, Top left, the 3.9 Å-resolution cryo-EM density map of RP2. Top right, cross-section of the structure, showing the DNA inside the RNAP cleft. Bottom, same views as above, but coloured by local resolution35.
Extended Data Fig. 6 Sample cryo-EM density.
Stereo views of cryo-EM density (blue mesh) and superimposed models. a, Protein–DNA interactions for template-strand DNA of RPo. b, Downstream single-strand–double-strand fork junction of RPo. The template-strand +1 position (transcription start site, T + 1; lemon green) is unpaired and positioned near the RNAP active-site Mg2+ (not visible in this view). c, Same view as b, but showing cryo-EM density and model for RP2. The T + 1 nucleotide (lemon green) is base-paired with the non-template strand and more than 30 Å away from the RNAP active-site Mg2+.
Extended Data Fig. 7 Downstream duplex DNA helical axes.
The RPo, RP2 and Cor–RP2 structures were superimposed and the nucleic acid backbones are shown (RPo, blue; RP2, green; Cor–RP2, magenta). The helical axes of the downstream duplex DNAs, determined using curves44, are shown as thick coloured lines. The RP2 and Cor–RP2 downstream duplexes are tilted by about 35° relative to RPo.
Extended Data Fig. 8 Cryo-EM of RNAP–σA–RbpA–Cor–us-fork.
a, Flow chart showing the image-processing pipeline for the cryo-EM data of RNAP–σA–RbpA–Cor–us-fork complexes, starting with 4,897 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). Movies were frame-aligned and summed using MotionCor229. CTF estimation for each micrograph was calculated with Gctf30. A representative micrograph is shown following processing by MotionCor2. Particles were autopicked from each micrograph with Gautomatch and then sorted by 2D classification using RELION33 to assess quality. The twelve highest-populated classes from the 2D classification are shown. The dataset contained 925,009 particles. A subset of particles was used to generate an ab initio map in cryoSPARC31. Using the low-pass-filtered (30 Å) ab initio map as a template, two rounds of 3D heterogeneous refinement were performed using cryoSPARC in a binomial-like fashion. One major class emerged, which was refined using cryoSPARC homogenous refinement and then sharpened for model building. b, Angular distribution calculated in cryoSPARC for particle projections. Heat map shows number of particles for each viewing angle. c, Gold-standard FSC41, calculated by comparing the two independently determined half maps from cryoSPARC. The dotted line represents the 0.143 FSC cut-off, which indicates a nominal resolution of 4.4 Å. d, RNAP clamp conformations. The RPo structure (Fig. 1c) was used as a reference to superimpose other structures via α-carbon atoms of the structural core module (Supplementary Table 2), revealing a common core RNAP structure (grey molecular surface) but with shifts in the clamp modules. The clamp modules are shown as backbone cartoons with cylindrical helices (RPo, blue; Cor–RbpA-holo, olive; RbpA-holo (6C05), cyan; open-clamp (6BZO), red). The angles of clamp opening are shown (relative to RPo at 0°).
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Boyaci, H., Chen, J., Jansen, R. et al. Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding. Nature 565, 382–385 (2019). https://doi.org/10.1038/s41586-018-0840-5
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DOI: https://doi.org/10.1038/s41586-018-0840-5
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