Article | Published:

Structure of paused transcription complex Pol II–DSIF–NELF

Naturevolume 560pages601606 (2018) | Download Citation


Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 Å resolution. The structure reveals a tilted DNA–RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible ‘tentacles’ that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Shilatifard, A., Conaway, R. C. & Conaway, J. W. The RNA polymerase II elongation complex. Annu. Rev. Biochem. 72, 693–715 (2003)

  2. 2.

    Bentley, D. L. & Groudine, M. A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. Nature 321, 702–706 (1986).

  3. 3.

    Kao, S.-Y., Calman, A. F., Luciw, P. A. & Peterlin, B. M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330, 489–493 (1987).

  4. 4.

    Gilmour, D. S. & Lis, J. T. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. Mol. Cell. Biol. 6, 3984–3989 (1986).

  5. 5.

    Strobl, L. J. & Eick, D. Hold back of RNA polymerase II at the transcription start site mediates down-regulation of c-myc in vivo. EMBO J. 11, 3307–3314 (1992).

  6. 6.

    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).

  7. 7.

    Yamaguchi, Y. et al. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97, 41–51 (1999).

  8. 8.

    Narita, T. et al. Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex. Mol. Cell. Biol. 23, 1863–1873 (2003).

  9. 9.

    Marshall, N. F. & Price, D. H. Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J. Biol. Chem. 270, 12335–12338 (1995).

  10. 10.

    Wei, P., Garber, M. E., Fang, S. M., Fischer, W. H. & Jones, K. A. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92, 451–462 (1998).

  11. 11.

    Fujinaga, K. et al. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol. Cell. Biol. 24, 787–795 (2004).

  12. 12.

    Yamada, T. et al. P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol. Cell 21, 227–237 (2006).

  13. 13.

    Marshall, N. F., Peng, J., Xie, Z. & Price, D. H. Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J. Biol. Chem. 271, 27176–27183 (1996).

  14. 14.

    Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).

  15. 15.

    Williams, L. H. et al. Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks. Mol. Cell 58, 311–322 (2015).

  16. 16.

    Adelman, K. et al. Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling. Proc. Natl Acad. Sci. USA 106, 18207–18212 (2009).

  17. 17.

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

  18. 18.

    Martinez-Rucobo, F. W. & Cramer, P. Structural basis of transcription elongation. Biochim. Biophys. Acta 1829, 9–19 (2013).

  19. 19.

    Bernecky, C., Plitzko, J. M. & 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).

  20. 20.

    Ehara, H. et al. Structure of the complete elongation complex of RNA polymerase II with basal factors. Science 357, 921–924 (2017).

  21. 21.

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

  22. 22.

    Rao, J. N. et al. Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription. Biochem. J. 400, 449–456 (2006).

  23. 23.

    Palangat, M., Meier, T. I., Keene, R. G. & Landick, R. Transcriptional pausing at +62 of the HIV-1 nascent RNA modulates formation of the TAR RNA structure. Mol. Cell 1, 1033–1042 (1998).

  24. 24.

    Larson, M. H. et al. A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science 344, 1042–1047 (2014).

  25. 25.

    Cheung, A. C. M. & Cramer, P. Structural basis of RNA polymerase II backtracking, arrest and reactivation. Nature 471, 249–253 (2011).

  26. 26.

    Cheung, A. C. M., Sainsbury, S. & Cramer, P. Structural basis of initial RNA polymerase II transcription. EMBO J. 30, 4755–4763 (2011).

  27. 27.

    Kang, J. Y. et al. RNA polymerase accommodates a pause RNA hairpin by global conformational rearrangements that prolong pausing. Mol. Cell 69, 802–815.e1 (2018).

  28. 28.

    Guo, X. et al. Structural basis for NusA stabilized transcriptional pausing. Mol. Cell 69, 816–827.e4 (2018).

  29. 29.

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

  30. 30.

    Zhang, G. et al. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell 98, 811–824 (1999).

  31. 31.

    Kettenberger, H., Armache, K.-J. & Cramer, P. Architecture of the RNA polymerase II–TFIIS complex and implications for mRNA cleavage. Cell 114, 347–357 (2003).

  32. 32.

    Wang, D., Bushnell, D. A., Westover, K. D., Kaplan, C. D. & Kornberg, R. D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127, 941–954 (2006).

  33. 33.

    Vassylyev, D. G., Vassylyeva, M. N., Perederina, A., Tahirov, T. H. & Artsimovitch, I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007).

  34. 34.

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

  35. 35.

    Yamaguchi, Y., Inukai, N., Narita, T., Wada, T. & Handa, H. Evidence that negative elongation factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA. Mol. Cell. Biol. 22, 2918–2927 (2002).

  36. 36.

    Pagano, J. M. et al. Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions. PLoS Genet. 10, e1004090 (2014).

  37. 37.

    Gressel, S. et al. CDK9-dependent RNA polymerase II pausing controls transcription initiation. eLife 6, e29736 (2017).

  38. 38.

    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).

  39. 39.

    Landick, R. The regulatory roles and mechanism of transcriptional pausing. Biochem. Soc. Trans. 34, 1062–1066 (2006).

  40. 40.

    Reines, D., Ghanouni, P., Li, Q. Q. & Mote, J., Jr. The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage. J. Biol. Chem. 267, 15516–15522 (1992).

  41. 41.

    Adelman, K. et al. Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell 17, 103–112 (2005).

  42. 42.

    Nechaev, S. et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335–338 (2010).

  43. 43.

    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).

  44. 44.

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

  45. 45.

    Bochkareva, A., Yuzenkova, Y., Tadigotla, V. R. & Zenkin, N. Factor-independent transcription pausing caused by recognition of the RNA–DNA hybrid sequence. EMBO J. 31, 630–639 (2012).

  46. 46.

    Imashimizu, M. et al. Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo. Genome Biol. 16, 98 (2015).

  47. 47.

    Kireeva, M. L. & Kashlev, M. Mechanism of sequence-specific pausing of bacterial RNA polymerase. Proc. Natl Acad. Sci. USA 106, 8900–8905 (2009).

  48. 48.

    Palangat, M., Hittinger, C. T. & Landick, R. Downstream DNA selectively affects a paused conformation of human RNA polymerase II. J. Mol. Biol. 341, 429–442 (2004).

  49. 49.

    Cheng, B. & Price, D. H. Properties of RNA polymerase II elongation complexes before and after the P-TEFb-mediated transition into productive elongation. J. Biol. Chem. 282, 21901–21912 (2007).

  50. 50.

    Vos, S. M. et al. Structure of activated transcription complex Pol II–DSIF–PAF–SPT6. Nature (2018).

  51. 51.

    Gradia, S. D. et al. MacroBac: new technologies for robust and efficient large-scale production of recombinant multiprotein complexes. Methods Enzymol. 592, 1–26 (2017).

  52. 52.

    Hu, X. et al. A mediator-responsive form of metazoan RNA polymerase II. Proc. Natl Acad. Sci. USA 103, 9506–9511 (2006).

  53. 53.

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

  54. 54.

    Xu, J. et al. Structural basis for the initiation of eukaryotic transcription-coupled DNA repair. Nature 551, 653–657 (2017).

  55. 55.

    Kireeva, M. L., Komissarova, N., Waugh, D. S. & Kashlev, M. The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex. J. Biol. Chem. 275, 6530–6536 (2000).

  56. 56.

    Komissarova, N., Kireeva, M. L., Becker, J., Sidorenkov, I. & Kashlev, M. Engineering of elongation complexes of bacterial and yeast RNA polymerases. Methods Enzymol. 371, 233–251 (2003).

  57. 57.

    Tegunov, D. & Cramer, P. Real-time cryo-EM data pre-processing with Warp. Preprint at (2018).

  58. 58.

    Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

  59. 59.

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

  60. 60.

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

  61. 61.

    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).

  62. 62.

    Fernandez-Leiro, R. & Scheres, S. H. W. A pipeline approach to single-particle processing in RELION. Acta Crystallogr. D 73, 496–502 (2017).

  63. 63.

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

  64. 64.

    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).

  65. 65.

    Trabuco, L. G., Villa, E., Mitra, K., Frank, J. & Schulten, K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673–683 (2008).

  66. 66.

    Adamczak, R., Porollo, A. & Meller, J. Combining prediction of secondary structure and solvent accessibility in proteins. Proteins 59, 467–475 (2005).

  67. 67.

    Buchan, D. W. A., Minneci, F., Nugent, T. C. O., Bryson, K. & Jones, D. T. Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–W357 (2013).

  68. 68.

    Kim, D. E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004).

  69. 69.

    Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

  70. 70.

    Sun, Q. et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proc. Natl Acad. Sci. USA 110, 1303–1308 (2013).

  71. 71.

    Andrade, M. A., Petosa, C., O’Donoghue, S. I., Müller, C. W. & Bork, P. Comparison of ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18 (2001).

  72. 72.

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

  73. 73.

    Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).

  74. 74.

    Combe, C. W., Fischer, L. & Rappsilber, J. xiNET: cross-link network maps with residue resolution. Mol. Cell. Proteomics 14, 1137–1147 (2015).

  75. 75.

    Kosinski, J. et al. Xlink Analyzer: software for analysis and visualization of cross-linking data in the context of three-dimensional structures. J. Struct. Biol. 189, 177–183 (2015).

  76. 76.

    Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

  77. 77.

    Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

Download references


We thank E. Wolf for pig thymus, F. Fischer and U. Neef for maintaining insect cell stocks, C. Oberthür and G. Kokic for assistance with protein purification, A. Linden and C.-T. Lee for help with crosslinking mass spectrometry, C. Bernecky for discussions and for sharing the DSIF plasmid before publication, and D. Tegunov and C. Wigge for electron microscopy support. S.M.V. was supported by an EMBO Long-Term Fellowship (ALTF 745-2014). H.U. was supported by the Deutsche Forschungsgemeinschaft (DFG SFB860). P.C. was supported by the Advanced Grant TRANSREGULON (grant agreement 693023) of the European Research Council, and the Volkswagen Foundation.

Reviewer information

Nature thanks K. Adelman, S. Darst and R. Landick for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Seychelle M. Vos, Lucas Farnung


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

    • Seychelle M. Vos
    • , Lucas Farnung
    •  & Patrick Cramer
  2. Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry, Göttingen, Germany

    • Henning Urlaub
  3. University Medical Center Göttingen, Institute of Clinical Chemistry, Bioanalytics Group, Göttingen, Germany

    • Henning Urlaub


  1. Search for Seychelle M. Vos in:

  2. Search for Lucas Farnung in:

  3. Search for Henning Urlaub in:

  4. Search for Patrick Cramer in:


S.M.V. designed and conducted all experiments, unless stated otherwise. L.F. collected and processed electron microscopy data. L.F. and S.M.V. built the model. H.U. performed mass spectrometry. P.C. supervised the project. S.M.V., L.F. and P.C. wrote the manuscript with input from H.U.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Patrick Cramer.

Extended data figures and tables

  1. Extended Data Fig. 1 Protein preparation and nucleic acid scaffold design.

    a, Quality of purified proteins used in this study. Purified proteins (0.9 µg) were run on 4–12% SDS–PAGE and stained with Coomassie blue. An asterisk demarcates SPT5 lacking an N-terminal region. b, Nucleic acid scaffold used for RNA extension assays, the ‘pause assay scaffold’. Template DNA is coloured in dark blue, non-template DNA is in light blue, and RNA is in red. c, Nucleic acid scaffold used for binding experiments and for cryo-EM analysis, the ‘HIV-1 pause scaffold’. Colours are the same as in b. d, SDS–PAGE analysis of fractions obtained from size-exclusion chromatography. The fractions used for cryo-EM analysis are marked. e, Quantification of the RNA extension assays shown in Fig. 1. The amount of elongated product was measured for each time point. Points are the mean of three independent experiments and error bars represent the standard deviation between experiments. f, Quantification of the RNA extension assays shown in Fig. 6. The amount of elongated product was measured for each time point. Points are the mean of three independent experiments and error bars represent the standard deviation between experiments.

  2. Extended Data Fig. 2 RNA extension assays on HIV-1 nucleic acid scaffold.

    a, HIV-1 nucleic acid scaffold used for RNA extension assays. The sequence is slightly altered from that used for cryo-EM to allow extension for eight bases before pausing. Known pause and arrest sites are marked on the sequence. b–e, Pol II ECs (75 nM) were reconstituted on the HIV-1 transcription scaffold (50 nM). A single reaction was incubated with ATP, CTP and UTP (0.5 mM) for 5 min to indicate the pause site (far right lane). Buffer (b), DSIF (b), NELF (c), DSIF and NELF (c), NELF tentacle mutants (d), or DSIF and NELF tentacle mutants (e) (300 nM) were incubated with the Pol II EC. NTPs were added (0.5 mM) and aliquots were taken at specific time points. Only a fraction of the starting RNA is successfully elongated owing to incomplete EC formation(see Methods for more information).

  3. Extended Data Fig. 3 Cryo-EM data collection and processing.

    a, Representative micrograph of data collection for the PEC, shown at a defocus of 2.5 µm. The micrograph is representative of 11,740 micrographs. b, Representative 2D classes of PEC particles. c, Classification tree for data processing. The numbers used to identify each map are shown above the corresponding map.

  4. Extended Data Fig. 4 Quality of cryo-EM data.

    a, b, Estimation of average resolution, showing global (a) and focused (b) refinement. The lines indicate the FSC between the half maps of the reconstruction. FSC curves are shown for each map. c–e, Angular distribution of particles from overall refinements and local resolution of selected refinements for the PEC (map 1) (c), NELF-A–NELF-C selected (map 2) (d) and NELF-B selected (map 3) (e). Shading from blue to yellow indicates the number of particles at a given orientation. Reconstructions coloured by local resolution. Shading from red to blue indicates the local resolution according to the accompanying colour gradient. Absolute values are indicated. B-factors were used as indicated.

  5. Extended Data Fig. 5 Fit of PEC structure in representative densities.

    a, PEC structure fit in electron density contoured to 6 Å from map 3. Front, top, and side views are shown. b–f, Electron density for various elements of the PEC structure shown as meshes. b, A loop connecting NELF-C helices 17 and 18 (map 3, grey mesh) contacts the trigger loop (map 2, lime-green mesh). c, NELF-B (map 3). d, NELF-C contacts the RPB1 funnel helices (α20, α21). e, Funnel helices (α20, α21). f, The NELF-A–NELF-C interaction (A-α6, C-α2′).

  6. Extended Data Fig. 6 Crosslinking mass spectrometry analysis.

    a, Overview of PEC crosslinks obtained with BS3. Subunits coloured as in Fig. 1. The thickness of the grey line connecting subunits signifies the number of crosslinks obtained between subunits. b, Histogram of unique crosslinks that were mapped onto our structure. Distances are measured between Cα pairs using Xlink analyser75 for crosslinks with a score greater than 5. The number of unique crosslinks detected at each distance is indicated. A dotted black line marks the 30 Å distance cut-off for BS3. c–e, Representative spectra from crosslinking mass spectrometry experiments. Blue, red and dark blue correspond to b-, y-, and a-ions of peptide A, respectively. Green, orange and dark green correspond to b-, y-, and a-ions of peptide B. Black bars drawn between lysines indicate crosslinking sites. Red highlighted ‘C’ represents carbamido-methylated cysteine residues. Relative intensity of m/z is plotted. Spectra are representative of one biological and two technical replicates.

  7. Extended Data Fig. 7 Comparison of previous structures to the PEC.

    a, The PEC and Pol II–DSIF EC structures were aligned by their Pol II cores. Slight differences are observed in DSIF bound to the PEC (green) in comparison to the Pol II–DSIF EC19 (yellow). b, The previously solved NELF-A–NELF-C dimerization crystal structure21 (PDB ID: 5L3X) and the NELF-A–NELF-C dimerization domain from the PEC cryo-EM structure were aligned on the NELF-C subunit. The NELF-A–NELF-C dimer widens when bound to Pol II (r.m.s.d. 1.39 Å). c, NELF-A tentacle crosslinks mapped onto the PEC. NELF-A and corresponding Pol II or DSIF residues are indicated. Related to Fig. 6. d, NELF-E tentacle crosslinks mapped onto the PEC. NELF-E and corresponding Pol II or DSIF residues are indicated. Related to Fig. 6.

  8. Extended Data Fig. 8 Conservation of Pol II and NELF elements.

    Sequence alignments were made using MAFFT76 and were visualized in Jalview77. Sequences elements are coloured by identity. Darker shades of blue indicate higher levels of identity. Red boxes demarcate the interacting residue. a, Conservation of RPB1 funnel helix and shelf module residues that interact with NELF-C. Organisms that encode for NELF are indicated. b, Conservation of NELF-C residues that interact with RPB1 funnel helix and shelf module residues. c, Conservation of NELF-C residues that interact with the RPB1 trigger loop. d, Conservation of Pol I (RPA1), Pol II (RPB1) and Pol III (RPC1) large subunits and putative NELF-C interaction interface.

  9. Extended Data Fig. 9 TFIIS does not interact with the PEC.

    a, Shelf movement relative to the Pol II core during reactivation. An arrested Pol II crystal structure (PDB ID: 3PO2) and the crystal structure of its reactivation intermediate (PDB ID: 3PO3) were aligned on their Pol II core modules25,31 (dark grey). The shelf module (pink) rotates away from the core module during reactivation. b, TFIIS does not bind the PEC. Fractions from size-exclusion chromatography with Pol II, DSIF, NELF and TFIIS. The EC was incubated with DSIF, NELF and TFIIS and applied to a Superose 6 column. The PEC is formed, but TFIIS does not migrate with the PEC. The experiment was performed twice. c, TFIIS binds the Pol II–DSIF EC. Fractions from size-exclusion chromatography with Pol II, DSIF and TFIIS. The EC was incubated with DSIF and TFIIS. A stable Pol II–DSIF–TFIIS EC is formed. The experiment was performed twice.

  10. Extended Data Table 1 Components of the PEC
  11. Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics
  12. Extended Data Table 3 RNA Pol II–NELF interactions

Supplementary information

  1. Supplementary Figure

    This file contains Supplementary Figure 1 Gel Source data 1. Uncropped gel scans. Size marker is indicated for purified protein gels. Dotted boxes indicate gel region used for figures. Source data for Figures 1a, b, 6c, and Extended Data Figures 1a, 2b-e, 9b, c.

  2. Reporting Summary

  3. Supplementary Tables

    This file contains Supplementary Tables 1-6 and a Supplementary Tables Guide.

  4. Supplementary Video 1 Overview of PEC structure.

    An overview of the PEC structure and corresponding cryo-EM density.

  5. Supplementary Video 2 Tilting of the DNA-RNA hybrid and NELF interaction.

    This video shows typical DNA-RNA hybrids found in pre- and post-translocated Pol II-DSIF ECs. It then shows conversion into the paused state with an unusual tilted RNA-DNA hybrid found in the PEC. NELF is then seen interacting with the complex, stabilizing the paused state.

  6. Supplementary Video 3 NELF-AC binds over the Pol II funnel.

    This video shows the location of the Pol II funnel. NELF is then docked to Pol II and covers the funnel region.

About this article

Publication history




Issue Date


Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.