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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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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