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Structure of paused transcription complex Pol II–DSIF–NELF

Abstract

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.

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Fig. 1: Formation of the paused Pol II–DSIF–NELF elongation complex.
Fig. 2: Cryo-EM structure of the PEC.
Fig. 3: Tilted DNA–RNA hybrid.
Fig. 4: NELF structure.
Fig. 5: NELF restricts Pol II mobility.
Fig. 6: NELF tentacles reach DSIF and RNA.

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Acknowledgements

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.

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Contributions

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.

Corresponding author

Correspondence to Patrick Cramer.

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The authors declare no competing interests.

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Extended data figures and tables

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.

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).

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.

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.

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′).

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.

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.

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.

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.

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

Supplementary information

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.

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Supplementary Tables

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

Supplementary Video 1 Overview of PEC structure.

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

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.

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.

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Vos, S.M., Farnung, L., Urlaub, H. et al. Structure of paused transcription complex Pol II–DSIF–NELF. Nature 560, 601–606 (2018). https://doi.org/10.1038/s41586-018-0442-2

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