Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
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Adelman, K. & Lis, J. T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).
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).
Yamaguchi, Y. et al. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97, 41–51 (1999).
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).
Ehara, H. et al. Structure of the complete elongation complex of RNA polymerase II with basal factors. Science 357, 921–924 (2017).
Vos, S. M., Farnung, L., Urlaub, H. & Cramer, P. Structure of paused transcription complex Pol II–DSIF–NELF. Nature https://doi.org/10.1038/s41586-018-0442-2 (2018).
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).
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).
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).
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).
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).
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).
Kim, J. B. & Sharp, P. A. Positive transcription elongation factor B phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently of cyclin-dependent kinase-activating kinase. J. Biol. Chem. 276, 12317–12323 (2001).
Yu, M. et al. RNA polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II. Science 350, 1383–1386 (2015).
Chen, F. X. et al. PAF1, a molecular regulator of promoter-proximal pausing by RNA polymerase II. Cell 162, 1003–1015 (2015).
Zhu, B. et al. The human PAF complex coordinates transcription with events downstream of RNA synthesis. Genes Dev. 19, 1668–1673 (2005).
Mueller, C. L. & Jaehning, J. A. Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex. Mol. Cell. Biol. 22, 1971–1980 (2002).
Krogan, N. J. et al. RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22, 6979–6992 (2002).
Kim, J., Guermah, M. & Roeder, R. G. The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS. Cell 140, 491–503 (2010).
Van Oss, S. B., Cucinotta, C. E. & Arndt, K. M. Emerging insights into the roles of the Paf1 complex in gene regulation. Trends Biochem. Sci. 42, 788–798 (2017).
Ardehali, M. B. et al. Spt6 enhances the elongation rate of RNA polymerase II in vivo. EMBO J. 28, 1067–1077 (2009).
Kaplan, C. D., Laprade, L. & Winston, F. Transcription elongation factors repress transcription initiation from cryptic sites. Science 301, 1096–1099 (2003).
Endoh, M. et al. Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. Mol. Cell. Biol. 24, 3324–3336 (2004).
Wada, T. et al. FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH. Mol. Cell 5, 1067–1072 (2000).
Kaplan, C. D., Holland, M. J. & Winston, F. Interaction between transcription elongation factors and mRNA 3′-end formation at the Saccharomyces cerevisiae GAL10-GAL7 locus. J. Biol. Chem. 280, 913–922 (2005).
Adelman, K. et al. Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26, 250–260 (2006).
Dronamraju, R. & Strahl, B. D. A feed forward circuit comprising Spt6, Ctk1 and PAF regulates Pol II CTD phosphorylation and transcription elongation. Nucleic Acids Res. 42, 870–881 (2014).
Xu, C. & Min, J. Structure and function of WD40 domain proteins. Protein Cell 2, 202–214 (2011).
Close, D. et al. Crystal structures of the S. cerevisiae Spt6 core and C-terminal tandem SH2 domain. J. Mol. Biol. 408, 697–713 (2011).
Edwards, A. M., Kane, C. M., Young, R. A. & Kornberg, R. D. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J. Biol. Chem. 266, 71–75 (1991).
Jasiak, A. J. et al. Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex. J. Biol. Chem. 283, 26423–26427 (2008).
Schulz, D., Pirkl, N., Lehmann, E. & Cramer, P. Rpb4 subunit functions mainly in mRNA synthesis by RNA polymerase II. J. Biol. Chem. 289, 17446–17452 (2014).
Swanson, M. S. & Winston, F. SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. Genetics 132, 325–336 (1992).
Hartzog, G. A., Wada, T., Handa, H. & Winston, F. Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 12, 357–369 (1998).
Qiu, H., Hu, C., Wong, C.-M. & Hinnebusch, A. G. The Spt4p subunit of yeast DSIF stimulates association of the Paf1 complex with elongating RNA polymerase II. Mol. Cell. Biol. 26, 3135–3148 (2006).
Squazzo, S. L. et al. The Paf1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO J. 21, 1764–1774 (2002).
Chen, Y. et al. DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation. Genes Dev. 23, 2765–2777 (2009).
Liu, Y. et al. Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of the PAF complex. Mol. Cell. Biol. 29, 4852–4863 (2009).
Zhou, K., Kuo, W.-H. W., Fillingham, J. & Greenblatt, J. F. Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5. Proc. Natl Acad. Sci. USA 106, 6956–6961 (2009).
He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016).
Turtola, M. & Belogurov, G. A. NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble. eLife 5, e18096 (2016).
Lu, X. et al. Multiple P-TEFbs cooperatively regulate the release of promoter-proximally paused RNA polymerase II. Nucleic Acids Res. 44, 6853–6867 (2016).
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).
Sdano, M. A. et al. A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription. eLife 6, e28723 (2017).
Yoh, S. M., Cho, H., Pickle, L., Evans, R. M. & Jones, K. A. The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev. 21, 160–174 (2007).
Yoh, S. M., Lucas, J. S. & Jones, K. A. The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. Genes Dev. 22, 3422–3434 (2008).
Battaglia, S. et al. RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases. eLife 6, e25637 (2017).
Kireeva, M. et al. RNA–DNA and DNA–DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate. Nucleic Acids Res. 46, 5764–5775 (2018).
Ng, H. H., Robert, F., Young, R. A. & Struhl, K. Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol. Cell 11, 709–719 (2003).
Bortvin, A. & Winston, F. Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Science 272, 1473–1476 (1996).
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).
Vos, S. M. et al. Architecture and RNA binding of the human negative elongation factor. eLife 5, e14981 (2016).
Kapust, R. B. & Waugh, D. S. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674 (1999).
Sydow, J. F. et al. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol. Cell 34, 710–721 (2009).
Sidorenkov, I., Komissarova, N. & Kashlev, M. Crucial role of the RNA:DNA hybrid in the processivity of transcription. Mol. Cell 2, 55–64 (1998).
Larson, M. H. et al. A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science 344, 1042–1047 (2014).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
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).
Xu, J. et al. Structural basis for the initiation of eukaryotic transcription-coupled DNA repair. Nature 551, 653–657 (2017).
Kireeva, M. L., Komissarova, N. & Kashlev, M. Overextended RNA:DNA hybrid as a negative regulator of RNA polymerase II processivity. J. Mol. Biol. 299, 325–335 (2000).
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).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
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).
Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
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).
Plaschka, C. et al. Architecture of the RNA polymerase II–Mediator core initiation complex. Nature 518, 376–380 (2015).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
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).
Afonine, P. V., Headd, J. J., Terwilliger, T. & Adams, P. D. New tool: phenix.real_space_refine. Comput. Crystallogr. Newsl. 4, 43–44 (2013).
Kim, D. E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004).
Halbach, F., Reichelt, P., Rode, M. & Conti, E. The yeast ski complex: crystal structure and RNA channeling to the exosome complex. Cell 154, 814–826 (2013).
Adamczak, R., Porollo, A. & Meller, J. Combining prediction of secondary structure and solvent accessibility in proteins. Proteins 59, 467–475 (2005).
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).
Karpenahalli, M. R., Lupas, A. N. & Söding, J. TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences. BMC Bioinformatics 8, 2 (2007).
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).
Xu, Y. et al. Architecture of the RNA polymerase II–Paf1C–TFIIS transcription elongation complex. Nat. Commun. 8, 15741 (2017).
Rozenblatt-Rosen, O. et al. The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol. Cell. Biol. 25, 612–620 (2005).
Yart, A. et al. The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol. Cell. Biol. 25, 5052–5060 (2005).
Amrich, C. G. et al. Cdc73 subunit of Paf1 complex contains C-terminal Ras-like domain that promotes association of Paf1 complex with chromatin. J. Biol. Chem. 287, 10863–10875 (2012).
Cao, Q.-F. et al. Characterization of the human transcription elongation factor Rtf1: evidence for nonoverlapping functions of Rtf1 and the Paf1 complex. Mol. Cell. Biol. 35, 3459–3470 (2015).
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Dolinsky, T. J. et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522–W525 (2007).
Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).
Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).
Combe, C. W., Fischer, L. & Rappsilber, J. xiNET: cross-link network maps with residue resolution. Mol. Cell. Proteomics 14, 1137–1147 (2015).
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).
Kiianitsa, K., Solinger, J. A. & Heyer, W.-D. NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal. Biochem. 321, 266–271 (2003).
Oellerich, T. et al. SLP-65 phosphorylation dynamics reveals a functional basis for signal integration by receptor-proximal adaptor proteins. Mol. Cell. Proteomics 8, 1738–1750 (2009).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Gnad, F. et al. PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol. 8, R250 (2007).
Hornbeck, P. V. et al. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 (2015).
Wessel, D. & Flügge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).
Giansanti, P., Tsiatsiani, L., Low, T. Y. & Heck, A. J. R. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nat. Protoc. 11, 993–1006 (2016).
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).
Fuchs, M. R. et al. D3, the new diffractometer for the macromolecular crystallography beamlines of the Swiss Light Source. J. Synchrotron Radiat. 21, 340–351 (2014).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Sun, M., Larivière, L., Dengl, S., Mayer, A. & Cramer, P. A tandem SH2 domain in transcription elongation factor Spt6 binds the phosphorylated RNA polymerase II C-terminal repeat domain (CTD). J. Biol. Chem. 285, 41597–41603 (2010).
Diebold, M.-L. et al. Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II. J. Biol. Chem. 285, 38389–38398 (2010).
Schilbach, S. et al. Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature 551, 204–209 (2017).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
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).
We thank A. Kühn and M. Raabe for identifying phosphorylation sites by mass spectrometry, 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, X. Liu and M. Ochmann for help with cloning and crystal refinement, H. S. Hillen and Swiss Light Source PXII for help with crystallographic data collection, and M. Geyer for sharing wild-type P-TEFb expression plasmids. 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.
Nature thanks K. Adelman, S. Darst and R. Landick for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Protein preparation and phosphorylation activity of P-TEFb and RNA extension assays.
a, Quality of purified proteins used in this study (0.9 µg protein per lane). All proteins were purified at least twice. The representative gel was run twice. The asterisk denotes a SPT5 N-terminal degradation product. b, Nucleic acid scaffold used for RNA extension assays, termed the modified pause scaffold. c, Nucleic acid scaffold used for analytical gel filtration and for cryo-EM analysis, termed the EC* scaffold. d, P-TEFb kinase activity using a coupled ATP/NADH assay. Bars correspond to the absolute change in absorbance at 340 nm as a function of time. Error bars represent the standard deviation between three individual experiments. Each bar corresponds to the mean of three individual experiments. e, P-TEFb (100 nM) was incubated with GST-RPB1 CTD for different amounts of time as indicated. Membranes were incubated with antibodies that recognize phospho-Ser2 (3E10), phospho-Ser5 (3E8), or the CTD (MABI0601). Similar experiments were performed at least three times for the wild-type enzyme. The western blot for the D149N mutant was performed once. f, Pol II (75 nM) was incubated with wild-type P-TEFb or P-TEFb(D149N) (100 nM) and DSIF and NELF (150 nM). Reactions were quenched at various time points after the addition of GTP and CTP (10 µM). The experiment was performed three times. g, Quantification of extended RNA products in f. Points are the mean of three individual experiments and error bars represent the standard deviation between replicates. Source data: Supplementary Table 8. h, Pol II (75 nM) was incubated with the modified pause scaffold (50 nM) (Extended Data Fig. 1b), wild-type (WT) P-TEFb or inactive P-TEFb(D149N) (100 nM) and ATP (1 mM) (all lanes), and DSIF and NELF (150 nM). PAF was titrated into the reactions. The reactions were quenched 2 min after the addition of CTP and GTP (10 µM). Positions for a consensus pausing site (+2) and extended RNA (+7) are marked. RNA extension is incomplete because only a fraction of Pol II molecules assemble on the scaffold. The experiment was performed twice. i, Quantification of extended RNA products in h. Points are the mean of two individual experiments and error bars represent the standard deviation between replicates. Source data: Supplementary Table 8. j, Pol II (75 nM) was incubated with DSIF and NELF (150 nM) and wild-type P-TEFb or P-TEFb(D149N) (100 nM). PAF and SPT6 were titrated into the reactions. Reactions were quenched 1 min after the addition of GTP and CTP (10 µM). The experiment was performed three times. k, Quantification of extended RNA products in j. Points are the mean of three individual experiments and error bars represent the standard deviation between replicates. Source data: Supplementary Table 8. l, Nucleic acid scaffold used for RNA extension assays, termed the EC* transcription scaffold. m, RNA extension assays performed on the EC* transcription scaffold (50 nM). Pol II (75 nM) was incubated with elongation factors (7.5–750 nM) (DSIF, PAF, SPT6), active P-TEFb or inactive P-TEFb(D149N) (100 nM) and 1 mM ATP for 15 min. Reactions were quenched 1 min after the addition of GTP, CTP and UTP. Experiments were performed three times. A large fraction of RNA primer remains owing to incomplete assembly of the elongation complex (see Methods for more details).
a, Quantification of extended RNA products in Fig. 1a. Points are the mean of three individual experiments and error bars represent the standard deviation between replicates. Source data: Supplementary Table 8. b, PAF, DSIF and SPT6 (23.7–750 nM) were titrated against Pol II (75 nM) and wild-type P-TEFb or P-TEFb(D149N). Reactions were quenched 1 min after the addition of GTP and CTP (10 µM). The experiment was performed three times. c, Quantification of extended RNA products in b. Points are the mean of three individual experiments and error bars represent the standard deviation between replicates. Source data: Supplementary Table 8. d, Elongation factors (75 nM) were incubated with P-TEFb (100 nM) and ATP (1 mM). Reactions were quenched after 0.6 min after the addition of GTP and CTP (10 µM). The experiment was performed three times. e, Quantification of extended RNA products in d. Points are the mean of three individual experiments and error bars represent the standard deviation between replicates. Source data: Supplementary Table 8. f–j, SDS–PAGE analysis of size-exclusion chromatography fractions. The Pol II elongation complex was formed on the EC* scaffold. All experiments were performed at least twice. f, DSIF; g, PAF; h, SPT6; i, Pol II elongation complex, DSIF, PAF, SPT6; j, Pol II elongation complex, DSIF, PAF, SPT6, P-TEFb and ATP. Fractions used for cryo-EM are indicated. k, NELF is released from Pol II when PAF, wild-type P-TEFb and ATP are present as assessed by size-exclusion chromatography. Curves from the PEC and the PEC plus PAF are shown as a reference. The Pol II elongation complex was formed on the EC* scaffold. Each experiment was performed at least twice. l, SDS–PAGE analysis of size-exclusion chromatography fractions from the formation of PEC with PAF, P-TEFb and ATP. The experiment was performed twice. m, SDS–PAGE analysis of size-exclusion chromatography fractions from the formation of PEC with PAF. The experiment was performed twice.
a, Representative micrograph of the EC* shown at a defocus of −2.5 µm. Representative of 20,198 micrographs. b, Representative 2D classes of EC* particles. c, Classification tree for data processing.
a, Estimate of average resolution. Lines indicate the Fourier shell correlation (FSC) between the half maps of the reconstruction. b, Angular distribution of particles from overall refinement. Red dots indicate the presence of at least one particle image within ±1°. c, Reconstructions of EC* as coloured by local resolution. The overall reconstruction is shown with B-factor-sharpened and non-sharpened maps. The globally refined maps E and H are shown as non-B-factor-sharpened maps.
a, EC* fit in electron density (map A) contoured to 12 Å. Black ovals indicate regions where electron density was weak. Map F and map H are shown to indicate the improvement after focused classification and refinement. b–f, Electron density for various elements of the EC* shown as grey mesh. b, CTR9 vertex and TPRs 18–19, map H. c, CTR9 trestle helix, map H. d, WDR61, map H. e, C terminus of LEO1 and upstream DNA, map G. f, tSH2 crystal structure, map F. g, Core of SPT6, map E.
a, Overview of crosslinks obtained with BS3 in EC*. Connecting line thickness signifies the number of crosslinks obtained between subunits. b, Histogram of unique crosslinks and distances between Cα pairs that were mapped onto our structure. A dotted black line marks the 30 Å distance cutoff for BS3. The Venn diagram compares unique crosslinks between two biological replicates. c–g, Crosslinks mapped onto the final model. Residues involved in crosslinks are shown as spheres. Coloured rods connecting residues signify permitted (blue) or non-permitted (red) crosslinking distances. c, WDR61 and CTR9. d, DSIF KOW1 and KOWx–KOW4 domains and SPT6. e, A C-terminal extension of LEO1, NGN and KOW1 domain of SPT5 and RPB2. f, SPT6 and Pol II. g, CTR9 and Pol II.
a, Cartoon model of human tSH2 crystal structure shown in two different views. b, Human SPT6 tSH2 is structurally similar to previously obtained SPT6 tSH2 structures from S. cerevisiae29 (PDB ID: 3PSJ) (hot pink), Candida glabrata103 (PDB ID: 3PJP) (grey), and Antonospora locustae104 (PDB ID: 2XP1) (peach). c, Surface charge representation of the human SPT6 tSH2. d, Representative electron density from the crystal structure of tSH2. 2Fo−Fc maps contoured at 2σ are shown for several regions of the tSH2 crystal structure. e, 15 Å low-pass-filtered map E. The C-terminal density of SPT6 extends to CTR9. f, Alternative view to that shown in Fig. 5b. Two P-TEFb phosphorylation sites are demarcated (T1525, T1540). The T1540 site was not observed in the yeast linker that was used for crystallization. The CTD linker is modelled.
a, WDR61 is anchored by the vertex and TPRs 13, 18 and 19. b, c, SPT6 binds to the C1–C3 sheets of RPB7. b, Surface representation of the association of SPT6 with the RPB4–RPB7 stalk (RPB4, red; RPB7, cyan). c, Book view of b. RPB4–RPB7 and SPT6 are coloured according to surface charge (blue, positive; red, negative). d, Comparison of initiation factor and elongation factor binding sites. The yeast preinitiation complex bound to core mediator (PIC-cMed)105 (PDB ID: 5OQM) was aligned with the EC* Pol II core. e, Model of RNA, CTD and CTR paths extending from the EC*.
a, S. scrofa Pol II CTD P-TEFb phosphorylations assessed by western blot using antibodies raised against phospho-Tyr1 (3D12), phospho-Ser2 (3E10), phospho-Ser5 (3E8) and phospho-Ser7 (4E12) or the RPB1 body (F12) or CTD (MABI0601). Experiments with the phospho-antibodies were performed twice. The RPB1 body and CTD antibody experiments were performed once. b, Phosphorylation sites as determined by mass spectrometry. The experiment was performed two or more times with each protein. The reported sites were found in at least two independent replicates. c–e, Representative mass spectra. f, Phosphorylations map to flexible regions of the EC*. Spheres and dotted lines represent two phosphorylations and flexible regions, respectively.
Extended Data Fig. 10 P-TEFb phosphorylates the CTD linker and SPT6 tSH2 required for association with EC*.
a, Sequence alignment of the CTD linker from various species generated in Mafft106 and visualized in Jalview107 (S. cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus laevis, Mus musculus and Homo sapiens). Blue columns represent regions sharing sequence identity. Orange boxes represent phosphorylation sites reported here or those obtained previously in yeast44. b–f, Representative MS2 spectra of P-TEFb phosphorylated CTD linker peptides. Spectra are representative of two biological replicates. RPB1 residues serine 1514 (b; precursor m/z 759.804, z = +2, corresponding RPB1 residues 1503–1517), threonine 1518 (c; precursor m/z 548.730, z = +2, RPB1 residues 1511–1520), threonine 1525 (d; precursor m/z 608.245, z = +2, RPB1 residues 1521–1531), threonine 1540 (e; precursor m/z 701.789, z = +2, RPB1 residues 1532–1546) as well as serine 1584 and serine 1590 (f; precursor m/z 580.708, z = +2, RPB1 residues 1582–1592) are phosphorylated by P-TEFb in vitro. The sequence of the corresponding phosphorylated chymotryptic precursor peptide is shown with all identified b-ions (blue) and y-ions (red). Asterisks indicate neutral loss of phosphoric acid (H3PO4, Δ97.98 Da), which is commonly observed for phosphoserine- and phosphothreonine-containing peptides upon HCD fragmentation. Additionally, peaks corresponding to neutral loss of ammonia (NH3, Δ17.03 Da) or water (H2O, Δ18.01 Da) are labelled in orange. g, Pulldowns performed with full-length SPT6 and SPT6 ∆tSH2 and MBP-RPB1 CTD constructs in the presence of wild-type P-TEFb or P-TEFb(D149N). The gel is representative of two independent experiments. h, Quality of purified SPT6 ∆tSH2 (1–1297) (0.9 µg). i, Time-course transcription assay with SPT6 ∆tSH2, PAF, DSIF (75 nM) and wild-type P-TEFb or P-TEFb(D149N). The gel is representative of three independent experiments. j, Size-exclusion chromatography experiment as performed in Extended Data Fig. 1. SPT6 ∆tSH2 does not stably associate with the EC*. The experiment was performed twice. k, Nucleic acid association with full-length SPT6. Binding to single-stranded DNA (cyan), double-stranded DNA (blue) or RNA (red) was assessed by fluorescence anisotropy. Error bars reflect the standard deviation between three experimental replicates. Points represent the mean of three experimental replicates.
This file contains Supplementary Figure 1: Gel Source data 1. Uncropped gel scans for Figure 1a, and Extended Data Figures 1a, e, f, h, j, m, and 2b, d, f, g, h, i, j. Size marker is indicated for gels with purified protein. Dotted boxes indicate gel region used for figures. This file also contains Supplementary Figure 2: Gel Source data 2. Uncropped scan with size marker indication for Extended Data Figures 2l, m, and 10g, h, j, i. Size marker is indicated for gels with purified proteins. Dotted boxes indicate gel region used for figures.
This file contains Supplementary Tables 1-8, and a Supplementary Tables Guide.
Overview of EC* structure. An overview of the EC* structure and corresponding cryo-EM densities.
NELF and PAF bind Pol II in a mutually exclusive manner. The PEC and EC* cryo-EM structures are overlaid on their cores to show that NELF and PAF associate with similar Pol II surfaces.
Conformational changes in Pol II and DSIF upon PAF and SPT6 binding. DSIF, the Pol II stalk, and upstream DNA adopt different conformations in EC* than in the PEC.
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Vos, S.M., Farnung, L., Boehning, M. et al. Structure of activated transcription complex Pol II–DSIF–PAF–SPT6. Nature 560, 607–612 (2018). https://doi.org/10.1038/s41586-018-0440-4
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