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Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding

Nature volume 565, pages 382–385 (2019)Cite this article

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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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Fig. 1: Structures of M. tuberculosis transcription initiation complexes with AP3 promoter DNA.
Fig. 2: Protein–transcription bubble interactions.
Fig. 3: FL2, Sw2 and clamp dynamics.
Fig. 4: Structural energetics of RPo formation.

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).

References

  1. Browning, D. F. & Busby, S. J. W. Local and global regulation of transcription initiation in bacteria. Nat. Rev. Microbiol. 14, 638–650 (2016).

    Article  CAS  Google Scholar 

  2. Nogales, E., Louder, R. K. & He, Y. Structural Insights into the eukaryotic transcription initiation machinery. Annu. Rev. Biophys. 46, 59–83 (2017).

    Article  CAS  Google Scholar 

  3. Plaschka, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016).

    Article  ADS  CAS  Google Scholar 

  4. Buc, H. & McClure, W. R. Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps. Biochemistry 24, 2712–2723 (1985).

    Article  CAS  Google Scholar 

  5. Roe, J. H., Burgess, R. R. & Record, M. T. Jr. Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the λPR promoter. J. Mol. Biol. 176, 495–522 (1984).

    Article  CAS  Google Scholar 

  6. Saecker, R. M. & Record, M. T. Jr & deHaseth, P. L. Mechanism of bacterial transcription initiation: RNA polymerase–promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J. Mol. Biol. 412, 754–771 (2011).

    Article  CAS  Google Scholar 

  7. Belogurov, G. A. et al. Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457, 332–335 (2008).

    Article  ADS  Google Scholar 

  8. Mukhopadhyay, J. et al. The RNA polymerase ‘switch region’ is a target for inhibitors. Cell 135, 295–307 (2008).

    Article  CAS  Google Scholar 

  9. Hubin, E. A. et al. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. eLife 6, e22520 (2017).

    Article  Google Scholar 

  10. Hubin, E. A., Lilic, M., Darst, S. A. & Campbell, E. A. Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures. Nat. Commun. 8, 16072 (2017).

    Article  ADS  CAS  Google Scholar 

  11. Lane, W. J. & Darst, S. A. Molecular evolution of multisubunit RNA polymerases: structural analysis. J. Mol. Biol. 395, 686–704 (2010).

    Article  CAS  Google Scholar 

  12. Stallings, C. L. et al. CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence. Cell 138, 146–159 (2009).

    Article  CAS  Google Scholar 

  13. Feklistov, A. et al. RNA polymerase motions during promoter melting. Science 356, 863–866 (2017).

    Article  ADS  CAS  Google Scholar 

  14. Schulz, S. et al. TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle. Proc. Natl Acad. Sci. USA 113, E1816–E1825 (2016).

    Article  CAS  Google Scholar 

  15. He, Y., Fang, J., Taatjes, D. J. & Nogales, E. Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013).

    Article  ADS  CAS  Google Scholar 

  16. Chakraborty, A. et al. Opening and closing of the bacterial RNA polymerase clamp. Science 337, 591–595 (2012).

    Article  ADS  CAS  Google Scholar 

  17. Boyaci, H. et al. Fidaxomicin jams Mycobacterium tuberculosis RNA polymerase motions needed for initiation via RbpA contacts. eLife 7, e34823 (2018).

    Article  Google Scholar 

  18. Zhou, Y. N. & Jin, D. J. The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like ‘stringent’ RNA polymerases in Escherichia coli. Proc. Natl Acad. Sci. USA 95, 2908–2913 (1998).

    Article  ADS  CAS  Google Scholar 

  19. Trautinger, B. W. & Lloyd, R. G. Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase. EMBO J. 21, 6944–6953 (2002).

    Article  CAS  Google Scholar 

  20. Vassylyev, D. G. et al. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417, 712–719 (2002).

    Article  ADS  CAS  Google Scholar 

  21. Kouzine, F. et al. Global regulation of promoter melting in naive lymphocytes. Cell 153, 988–999 (2013).

    Article  CAS  Google Scholar 

  22. Tomko, E. J., Fishburn, J., Hahn, S. & Galburt, E. A. TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning. Nat. Struct. Mol. Biol. 24, 1139–1145 (2017).

    Article  CAS  Google Scholar 

  23. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    Article  Google Scholar 

  24. Srivastava, D. B. et al. Structure and function of CarD, an essential mycobacterial transcription factor. Proc. Natl Acad. Sci. USA 110, 12619–12624 (2013).

    Article  ADS  CAS  Google Scholar 

  25. Gonzalez-y-Merchand, J. A., Colston, M. J. & Cox, R. A. The rRNA operons of Mycobacterium smegmatis and Mycobacterium tuberculosis: comparison of promoter elements and of neighbouring upstream genes. Microbiology 142, 667–674 (1996).

    Article  CAS  Google Scholar 

  26. Davis, E., Chen, J., Leon, K., Darst, S. A. & Campbell, E. A. Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD. Nucleic Acids Res. 43, 433–445 (2015).

    Article  CAS  Google Scholar 

  27. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  28. Nicholson, W. V., White, H. & Trinick, J. An approach to automated acquisition of cryoEM images from lacey carbon grids. J. Struct. Biol. 172, 395–399 (2010).

    Article  CAS  Google Scholar 

  29. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  30. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  ADS  CAS  Google Scholar 

  31. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  Google Scholar 

  32. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  33. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  Google Scholar 

  34. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  Google Scholar 

  35. Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013).

    Article  Google Scholar 

  36. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  37. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  39. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  40. Irschik, H., Jansen, R., Höfle, G., Gerth, K. & Reichenbach, H. The corallopyronins, new inhibitors of bacterial RNA synthesis from Myxobacteria. J. Antibiot. 38, 145–152 (1985).

    Article  CAS  Google Scholar 

  41. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  Google Scholar 

  42. Bae, B., Feklistov, A., Lass-Napiorkowska, A., Landick, R. & Darst, S. A. Structure of a bacterial RNA polymerase holoenzyme open promoter complex. eLife 4, e08504 (2015).

    Article  Google Scholar 

  43. Bae, B. et al. CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex. eLife 4, e08505 (2015).

    Article  Google Scholar 

  44. Lavery, R. & Sklenar, H. Defining the structure of irregular nucleic acids: conventions and principles. J. Biomol. Struct. Dyn. 6, 655–667 (1989).

    Article  CAS  Google Scholar 

Download references

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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Authors and Affiliations

  1. Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA

    Hande Boyaci, James Chen, Seth A. Darst & Elizabeth A. Campbell

  2. Department of Microbial Drugs, Helmholtz Centre for Infection Research, Braunschweig, Germany

    Rolf Jansen

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  1. Hande Boyaci
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  2. James Chen
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  4. Seth A. Darst
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Contributions

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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Correspondence to Elizabeth A. Campbell.

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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°).

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table
Extended Data Table 2 Structural superpositions
Full size table

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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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