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Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp

Nature Structural & Molecular Biology volume 24, pages 809–815 (2017)Cite this article

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Abstract

During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II–DSIF elongation complex at a nominal resolution of 3.4 Å. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.

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Figure 1: Structure of the Pol II–DSIF EC.
Figure 2: Modeling of the Pol II–DSIF EC through a combination of cryo-EM and X-ray analysis.
Figure 3: Features of the DSIF DNA clamp.
Figure 4: Features of the DSIF RNA clamp.

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References

  1. Kwak, H. & Lis, J.T. Control of transcriptional elongation. Annu. Rev. Genet. 47, 483–508 (2013).

    Article  CAS  Google Scholar 

  2. Wada, T. et al. DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12, 343–356 (1998).

    Article  CAS  Google Scholar 

  3. Hartzog, G.A. & Fu, J. The Spt4-Spt5 complex: a multi-faceted regulator of transcription elongation. Biochim. Biophys. Acta 1829, 105–115 (2013).

    Article  CAS  Google Scholar 

  4. Yamaguchi, Y., Shibata, H. & Handa, H. Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond. Biochim. Biophys. Acta 1829, 98–104 (2013).

    Article  CAS  Google Scholar 

  5. NandyMazumdar, M. & Artsimovitch, I. Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins: shifting shapes and paradigms. BioEssays 37, 324–334 (2015).

    Article  CAS  Google Scholar 

  6. Werner, F. A nexus for gene expression-molecular mechanisms of Spt5 and NusG in the three domains of life. J. Mol. Biol. 417, 13–27 (2012).

    Article  CAS  Google Scholar 

  7. Shetty, A. et al. Spt5 plays vital roles in the control of sense and antisense transcription elongation. Mol. Cell 66, 77–88.e5 (2017).

    Article  CAS  Google Scholar 

  8. Lidschreiber, M., Leike, K. & Cramer, P. Cap completion and C-terminal repeat domain kinase recruitment underlie the initiation-elongation transition of RNA polymerase II. Mol. Cell. Biol. 33, 3805–3816 (2013).

    Article  CAS  Google Scholar 

  9. Wen, Y. & Shatkin, A.J. Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev. 13, 1774–1779 (1999).

    Article  CAS  Google Scholar 

  10. Wu, C.H. et al. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17, 1402–1414 (2003).

    Article  CAS  Google Scholar 

  11. Hirtreiter, A. et al. Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif. Nucleic Acids Res. 38, 4040–4051 (2010).

    Article  CAS  Google Scholar 

  12. Liu, B. & Steitz, T.A. Structural insights into NusG regulating transcription elongation. Nucleic Acids Res. 45, 968–974 (2017).

    Article  CAS  Google Scholar 

  13. Cheng, B. & Price, D.H. Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay. Nucleic Acids Res. 36, e135 (2008).

    Article  Google Scholar 

  14. Crickard, J.B., Fu, J. & Reese, J.C. Biochemical analysis of yeast suppressor of Ty 4/5 (Spt4/5) reveals the importance of nucleic acid interactions in the prevention of RNA polymerase II arrest. J. Biol. Chem. 291, 9853–9870 (2016).

    Article  CAS  Google Scholar 

  15. Missra, A. & Gilmour, D.S. Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex. Proc. Natl. Acad. Sci. USA 107, 11301–11306 (2010).

    Article  CAS  Google Scholar 

  16. Palangat, M., Renner, D.B., Price, D.H. & Landick, R. A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS. Proc. Natl. Acad. Sci. USA 102, 15036–15041 (2005).

    Article  CAS  Google Scholar 

  17. Baejen, C. Genome-wide analysis of RNA Polymerase II termination at protein-coding genes. Mol. Cell 66, 38–49 e6 (2017).

    Article  CAS  Google Scholar 

  18. Mayer, A. et al. The spt5 C-terminal region recruits yeast 3′ RNA cleavage factor I. Mol. Cell. Biol. 32, 1321–1331 (2012).

    Article  CAS  Google Scholar 

  19. Ponting, C.P. Novel domains and orthologues of eukaryotic transcription elongation factors. Nucleic Acids Res. 30, 3643–3652 (2002).

    Article  CAS  Google Scholar 

  20. Kyrpides, N.C., Woese, C.R. & Ouzounis, C.A. KOW: a novel motif linking a bacterial transcription factor with ribosomal proteins. Trends Biochem. Sci. 21, 425–426 (1996).

    Article  CAS  Google Scholar 

  21. Guo, M. et al. Core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation. Structure 16, 1649–1658 (2008).

    Article  CAS  Google Scholar 

  22. Klein, B.J. et al. RNA polymerase and transcription elongation factor Spt4/5 complex structure. Proc. Natl. Acad. Sci. USA 108, 546–550 (2011).

    Article  CAS  Google Scholar 

  23. Martinez-Rucobo, F.W., Sainsbury, S., Cheung, A.C. & Cramer, P. Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity. EMBO J. 30, 1302–1310 (2011).

    Article  CAS  Google Scholar 

  24. Li, W., Giles, C. & Li, S. Insights into how Spt5 functions in transcription elongation and repressing transcription coupled DNA repair. Nucleic Acids Res. 42, 7069–7083 (2014).

    Article  CAS  Google Scholar 

  25. Bernecky, C., Herzog, F., Baumeister, W., Plitzko, J.M. & Cramer, P. Structure of transcribing mammalian RNA polymerase II. Nature 529, 551–554 (2016).

    Article  CAS  Google Scholar 

  26. Wenzel, S., Martins, B.M., Rösch, P. & Wöhrl, B.M. Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface. Biochem. J. 425, 373–380 (2010).

    Article  CAS  Google Scholar 

  27. Meyer, P.A. et al. Structures and functions of the multiple KOW domains of transcription elongation factor Spt5. Mol. Cell. Biol. 35, 3354–3369 (2015).

    Article  CAS  Google Scholar 

  28. Barnes, C.O. et al. Crystal structure of a transcribing RNA polymerase II complex reveals a complete transcription bubble. Mol. Cell 59, 258–269 (2015).

    Article  CAS  Google Scholar 

  29. Qiu, Y. & Gilmour, D.S. Identification of regions in the Spt5 subunit of DRB sensitivity-inducing factor (DSIF) that are involved in promoter-proximal pausing. J. Biol. Chem. 292, 5555–5570 (2017).

    Article  CAS  Google Scholar 

  30. Buchan, D.W., Minneci, F., Nugent, T.C., Bryson, K. & Jones, D.T. Scalable web services for the PSIPRED protein analysis workbench. Nucleic Acids Res. 41, W349–W357 (2013).

    Article  Google Scholar 

  31. Jennings, B.H. et al. Locus-specific requirements for Spt5 in transcriptional activation and repression in Drosophila. Curr. Biol. 14, 1680–1684 (2004).

    Article  CAS  Google Scholar 

  32. Guo, S. et al. A regulator of transcriptional elongation controls vertebrate neuronal development. Nature 408, 366–369 (2000).

    Article  CAS  Google Scholar 

  33. Weixlbaumer, A., Leon, K., Landick, R. & Darst, S.A. Structural basis of transcriptional pausing in bacteria. Cell 152, 431–441 (2013).

    Article  CAS  Google Scholar 

  34. Turtola, M. & Belogurov, G.A. NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble. eLife 5, e18096 (2016).

    Article  Google Scholar 

  35. Kwak, Y.T. et al. Methylation of SPT5 regulates its interaction with RNA polymerase II and transcriptional elongation properties. Mol. Cell 11, 1055–1066 (2003).

    Article  CAS  Google Scholar 

  36. Proudfoot, N.J. Transcriptional termination in mammals: stopping the RNA polymerase II juggernaut. Science 352, aad9926 (2016).

    Article  Google Scholar 

  37. Cramer, P., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863–1876 (2001).

    Article  CAS  Google Scholar 

  38. Engel, C., Sainsbury, S., Cheung, A.C., Kostrewa, D. & Cramer, P. RNA polymerase I structure and transcription regulation. Nature 502, 650–655 (2013).

    Article  CAS  Google Scholar 

  39. Sekine, S., Murayama, Y., Svetlov, V., Nudler, E. & Yokoyama, S. The ratcheted and ratchetable structural states of RNA polymerase underlie multiple transcriptional functions. Mol. Cell 57, 408–421 (2015).

    Article  CAS  Google Scholar 

  40. Tagami, S. et al. Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein. Nature 468, 978–982 (2010).

    Article  CAS  Google Scholar 

  41. Sansó, M. et al. P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates. Genes Dev. 30, 117–131 (2016).

    Article  Google Scholar 

  42. He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016).

    Article  CAS  Google Scholar 

  43. Grohmann, D. et al. The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation. Mol. Cell 43, 263–274 (2011).

    Article  CAS  Google Scholar 

  44. Pei, Y. & Shuman, S. Interactions between fission yeast mRNA capping enzymes and elongation factor Spt5. J. Biol. Chem. 277, 19639–19648 (2002).

    Article  CAS  Google Scholar 

  45. Ehara, H. et al. Structure of the complete elongation complex of RNA polymerase II with basal factors. http://dx.doi.org/10.1126/science.aan8552 (2017).

  46. Vos, S.M. et al. Architecture and RNA binding of the human negative elongation factor. eLife 5, e14981 (2016).

    Article  Google Scholar 

  47. Korinek, A., Beck, F., Baumeister, W., Nickell, S. & Plitzko, J.M. Computer controlled cryo-electron microscopy: TOM2 a software package for high-throughput applications. J. Struct. Biol. 175, 394–405 (2011).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  51. Danev, R., Tegunov, D. & Baumeister, W. Using the Volta phase plate with defocus for cryo-EM single particle analysis. bioRxiv e23006 (2016).

  52. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  53. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  Google Scholar 

  54. Sheldrick, G.M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 479–485 (2010).

    Article  CAS  Google Scholar 

  55. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  57. Meka, H., Werner, F., Cordell, S.C., Onesti, S. & Brick, P. Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II. Nucleic Acids Res. 33, 6435–6444 (2005).

    Article  CAS  Google Scholar 

  58. 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 

  59. Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 47, 5.6 (2014).

    Article  Google Scholar 

  60. Tyka, M.D. et al. Alternate states of proteins revealed by detailed energy landscape mapping. J. Mol. Biol. 405, 607–618 (2011).

    Article  CAS  Google Scholar 

  61. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  62. Barad, B.A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  Google Scholar 

  63. Plaschka, C. et al. Architecture of the RNA polymerase II-mediator core initiation complex. Nature 518, 376–380 (2015).

    Article  CAS  Google Scholar 

  64. Sanner, M.F., Olson, A.J. & Spehner, J.C. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C. Plaschka and D. Tegunov for helpful discussions regarding EM, H. Hillen for assistance with X-ray data collection and phasing, H. Hillen, C. Dienemann, S. Sainsbury, C. Engel, S. Neyer, Y. Xu, and other members of the laboratory of P.C. for helpful discussions regarding X-ray crystallography, S. Neyer, C. Dienemann, and D. Tegunov for assistance with EM and X-ray validation figures, J. Wawrzinek for setting initial crystallization screens, S. Bilakovic for the modified pET-DUET-1 vector, J. Kellermann for N-terminal sequencing, and other members of the laboratory of P.C. for help and discussions. Part of this work was performed at the Swiss Light Source (SLS) at the Paul Scherrer Institut. P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935), the Advanced Grant 'TRANSREGULON' from the European Research Council (grant agreement no. 693023), and the Volkswagen Foundation.

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Authors and Affiliations

  1. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Carrie Bernecky & Patrick Cramer

  2. Department of Molecular Structural Biology, Max Planck Institute for Biochemistry, Martinsried, Germany

    Jürgen M Plitzko

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  1. Carrie Bernecky
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  2. Jürgen M Plitzko
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Contributions

C.B. designed and carried out experiments and data analysis. J.M.P. provided access to a high-end EM facility and provided advice on microscope setup. P.C. supervised research. C.B. and P.C. prepared the manuscript.

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Correspondence to Patrick Cramer.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM analysis of the Pol II–DSIF EC.

(a) SDS-PAGE analysis of the Pol II-DSIF-NELF EC after size exclusion chromatography. (b) Representative micrograph at −1.6 μm defocus. Scale bar, 100 nm. (c) Representative 2D classes from unsupervised classification. (d) Processing tree indicating 3D classification steps used to generate reconstructions with improved density for different DSIF domains. Densities from focused classifications are shown over a gray outline of the Pol II surface to provide context. Particle classes shown over a colored background were subjected to 3D refinement. Refined densities are shown next to the respective 3D classification. Coloring is as follows: DNA, blue; RPB4-RPB7 stalk domain, dark gray; RNA, red; DSIF domains as in Fig. 1.

Supplementary Figure 2 Cryo-EM reconstruction statistics.

Angular distribution plot for the DSIF-EC1 reconstruction. Color scale indicates number of particles matching a particular view. (b) Fourier shell correlation plots for the five DSIF-EC maps described in Table S1, as well as the model versus map correlations for the selected regions of the model and DSIF-EC1 or DSIF-EC2 densities (Methods). Magnified view to the right. (c) DSIF-EC1 map filtered to 15 Å with domains colored as in Fig. 1. Density for all DSIF domains that fold along the EC are visible at low resolution. (d-h) Local resolution estimates for the five DSIF-EC maps described in Table S1. All maps are shown in a top view. (d) DSIF-EC1, filtered to the nominal resolution of 3.4 Å and B-factor sharpened. (e) DSIF-EC2 shown filtered to 4 Å and B-factor sharpened, and a second time filtered to 5 Å to allow visualization of lower resolution domains. (f) DSIF-EC3, (g) DSIF-EC4, and (h) DSIF-EC5 filtered by local resolution.

Supplementary Figure 3 Cryo-EM and X-ray modeling and density fit of the Pol II–DSIF EC and the KOW6–7 domain.

(a) Density overlays of representative regions within the Pol II-DSIF EC and X-ray structures. RPB2 and KOW5, DSIF-EC1 map (3.4 Å); KOW3 and KOWx, DSIF-EC2 map (4 Å), KOWx-4 and KOW6-7, experimental density maps from SAD phasing in SHELX. (b) Two views of the KOW6-7 crystal structure with mutations leading to developmental defects shown as spheres; black, mutation identified in D. melanogaster; gray, mutation identified in D. rerio.

Supplementary Figure 4 Details of the DSIF DNA clamp.

(a) Comparison of the mammalian DSIF-EC with the archaeal (P. furiosus) clamp domain-NGN-Spt4 X-ray structure (PDB 3QQC)23. The archaeal clamp domain (dark gray) was superimposed on the mammalian clamp domain (light gray). Human DSIF coloring is as in Fig. 1. Archaeal Spt5 and Spt4 are shown in light and dark purple, respectively. The archaeal structure is shown as semi-transparent ribbon. Relative shift and rotation of the archaeal NGN-Spt4 is indicated. (b) Zoomed-in top view showing the density for the unmodeled RPB2 protrusion tip, which contacts helix α1 of the NGN domain. (c) Zoomed-in back view showing the density for the positively charged linker within the KOW1-L1 domain that contacts the RPB2 wall.

Supplementary Figure 5 Mutations in DSIF regions that contact nucleic acids are important for the binding of DSIF to the Pol II EC.

(a) Location of the mutated sites in DSIF shown as black spheres on the Pol II-DSIF EC structure colored as in Fig. 1. (b) SDS-PAGE analysis of equimolar amounts of purified DSIF mutants. (c) EMSA of EC20 with DSIF and the indicated mutant forms of DSIF. Elongation complexes were formed in vitro using a DNA-RNA bubble scaffold containing 20 nucleotides of 5’-FAM-labeled RNA (EC20) (Methods). RNA concentrations were 100 nM. Image was cropped to show only relevant areas.

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Supplementary Text and Figures

Supplementary Figures 1–5. (PDF 1055 kb)

Life Sciences Reporting Summary (PDF 138 kb)

Three-dimensional view of the Pol II-DSIF EC.

A movie showing a 360° rotation of the DSIF-EC model, as well as zoom-ins of the DNA and RNA clamp. Coloring is as in Fig. 1. (MP4 21030 kb)

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Bernecky, C., Plitzko, J. & Cramer, P. Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp. Nat Struct Mol Biol 24, 809–815 (2017). https://doi.org/10.1038/nsmb.3465

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