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Structure of complete Pol II–DSIF–PAF–SPT6 transcription complex reveals RTF1 allosteric activation

Abstract

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal ‘fastener’ helix retains PAF and is followed by a ‘latch’ that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.

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Fig. 1: RTF1 strongly stimulates Pol II elongation activity.
Fig. 2: Domain architecture and cryo-EM structure of the complete EC*.
Fig. 3: Structure of RTF1 and its interactions with Pol II.
Fig. 4: RTF1 interactions with DSIF and PAF.
Fig. 5: RTF1 latch stimulates elongation activity.
Fig. 6: Complete model of human PAF.
Fig. 7: Model of potential EC* interactions with chromatin-modifying enzymes.

Data availability

The electron density reconstructions and final model were deposited with the Electron Microscopy Data Base under accession code nos. EMD-1048010489, and with the Protein Data Bank under accession code PDB 6TED. Source data for Figs. 1, 4 and 5 are available with the paper online.

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Acknowledgements

We thank past and present members of the Cramer and Urlaub laboratories for their support and input and A. Sawicka for providing human cDNA. We thank R. Landick for discussions. S.M.V. was supported by an EMBO Long-Term Fellowship (no. ALTF 745-2014). H.U. was supported by the Deutsche Forschungsgemeinschaft (grant no. DFG SFB860). P.C. was supported by the Deutsche Forschungsgemeinschaft (grant nos. SFB860 and EXC 2067/1-390729940) and the Advanced Grant TRANSREGULON (grant no. 693023) of the European Research Council and the Volkswagen Foundation.

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S.M.V. conceived, designed and executed all experiments unless stated otherwise. S.M.V. and L.F. collected cryo-EM data. L.F. performed initial data processing and experiments with the RTF1-6A variant. A.L. and H.U. collected and analyzed crosslinking mass spectrometry data. S.M.V. and P.C. interpreted the data and wrote the manuscript with input from all authors. P.C. supervised research.

Corresponding author

Correspondence to Patrick Cramer.

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The authors declare no competing interests.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1

a, Quality of purified proteins used in this study. Purified proteins (1.1 µg per lane) were run on 4–12% SDS-PAGE and stained with Coomassie blue. b, Modified JUNB nucleic acid scaffold used for transcription assays. c–e, RNA extension assays on modified JUNB scaffold. Pol II (75 nM) was incubated with WT P-TEFb (100 nM) and 1 mM ATP for 15 min and indicated elongation factors (75 nM) at 30 °C. Aliquots were taken after GTP, CTP, and UTP addition at the indicated times. RNA primer and fully extended product are marked. c, (left) Pol II, (right) Pol II, DSIF, RTF1 d, (left) Pol II, DSIF, PAF, (right) Pol II, DSIF, PAF, SPT6 (EC*) e, (left) Pol II, DSIF, PAF, RTF1, (right) Pol II, DSIF, PAF, SPT6, RTF1. All experiments were performed at least 3 times. f–j, RNA extension assays as performed in C-E, except samples were incubated with WT (left) or D149N P-TEFb (right). RNA primer and fully extended product are indicated. f, Pol II, g, Pol II, DSIF, PAF, h, Pol II, DSIF, PAF, RTF1, i, Pol II, DSIF, PAF, SPT6, j, Pol II, DSIF, PAF, SPT6, RTF1. All experiments were performed at least 2 times. k, Quantification of extended product from 3 independent experiments. Points are the mean of the intensity of the extended product. Error bars represent mean ±s.d. l, RNA extension assay performed with increasing concentrations of RTF1 and constant concentrations (75 nM) of Pol II and DSIF. The reaction was treated as in C-E and quenched 1 min after NTP addition. This experiment was repeated 3 times.

Extended Data Fig. 2

a, Formation of complete EC* by size exclusion chromatography. b, EC*+RTF1 complex formation. SDS-PAGE analysis of size exclusion chromatography fractions. Fractions used for cryo-EM analysis are indicated. This experiment was performed at least n=5 times.

Extended Data Fig. 3 Quality of cryo-EM data.

a, Estimate of average resolution. The lines indicate the Fourier shell correlation (FSC) between the half maps of the reconstruction. FSC curves are shown for each deposited map. b, Angular distribution of particles from overall refinement (Map 1). Shading from blue to yellow indicates the number of particles at a given orientation. c, Complete EC* reconstructions as colored by local resolution as implemented in RELION 3.0. The overall refinement (Map 1) was subjected to local resolution calculations with an estimated B factor of −115 and −20. A B factor of −20 was applied to Map 2. Top and side views are shown.

Extended Data Fig. 4 Cryo-EM data collection and processing.

a, Representative denoised micrograph of complete EC* shown at a defocus of 2.5 µm. The scale bar has a length = 100 nm. b, Representative 2D classes of the refined model. The scale bar = 10 nm. c, Classification tree for data processing. Names used to identify each map are shown above the corresponding classification branch.

Extended Data Fig. 5 Crosslinking-MS analysis.

a, Overview of crosslinks obtained with BS3 in the complete EC*. Subunits colored as in Fig. 2. 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 analyzer78. The number of unique crosslinks detected at each distance is indicated. A dotted green line demarcates the 30 Å distance cutoff for BS3. Source data for this panel are provided in Supplementary Table 1. c, Crosslinks mapped onto our final model of RTF1 on Pol II using Xlink analyzer78. Residues involved in crosslinks are shown as spheres. Distances that are permitted by BS3 are shown in blue. Distances that exceed 30 Å are shown in red. d, The C-terminus of RTF1 interacts with CTR9 TPRs 5, 6, 7, and 11. Pink spheres on CTR9 indicate positions where RTF1 crosslinks with CTR9 were detected. Interacting residues are indicated.

Extended Data Fig. 6 Representative spectra from crosslinking MS experiments.

Blue and red correspond to b- and y- ions of peptide A, respectively. Green and orange correspond to b- and y- ions of peptide B. Black bars drawn between lysines indicate crosslinking sites. Relative intensity of m/z is plotted. The sequence of the detected peptide with the charge of the cross-link are displayed. Carbamidomethylated cysteines are colored red. a, RPB12 11, RTF1 486 b, RPB3 152, RTF1 555 c, RPB10 67, RTF1 504 d, PAF1 68, RTF1 649.

Extended Data Fig. 7 Fits of complete EC* in representative densities.

a, Fit of complete EC*-RTF1 in composite map used for refinement. Side, back and top views are shown as in Fig. 2. b, Complete EC* active site modelled in composite map used for refinement. Pol II active site is not altered upon RTF1 binding. Active site adopts post-translocated conformation. c, Fit of RTF1 helices and latch in local resolution filtered version of Map 2. d, RTF1 latch contacts with the RPB1 bridge helix and funnel helices in local resolution filtered version of Map 2. e, Fit of improved CTR9, WDR61, PAF1, and CDC73 model in Map 4. f, Fit of improved CTR9, WDR61, PAF1, and CDC73 model in Map 6. g, Fit of improved CTR9, WDR61, PAF1, and CDC73 model into previous CTR9 middle map H10.

Extended Data Fig. 8 S. cerevisiae Rpb2 loop occupies same region as RTF1 fastener extension.

a, The complete EC* structure, an elongating S. cerevisiae Pol II structure (PDB ID 3HOV)46, and a structure of E. coli RNA polymerase (PDB ID 4LJZ)79 were aligned on their cores. An extended loop in External 1 of S. cerevisiae Rpb2 and a hairpin in the β subunit of E. coli RNA polymerase occupy a similar region as the RTF1 latch adjacent to the funnel helices. b, Sequence alignment of the extended loop in S. cerevisiae Rpb2 External 1 that overlaps with the RTF1 binding site adjacent to the bridge helix. Darker shades of blue indicate higher levels of sequence conservation. The extended loop in Rpb2 external 1 appears to be absent in higher eukaryotes. Conserved S. cerevisiae Rpb2 Arg residues that lie in similar position to Arg residues found in RTF1 are marked with green boxes.

Extended Data Fig. 9 The effect of RTF1 truncations on RTF1 dependent stimulation of EC*.

Representative gel images for Fig. 5b. RTF1 truncation mutants were purified and tested for their ability to stimulate Pol II transcription in the context of complete EC*. Aliquots were taken at the indicated times and visualized by denaturing gel electrophoresis. The initial RNA primer band and the extended product band are indicated. Experiments were performed at least three separate times at 30 °C. a, Complete EC* with WT RTF1. b, Complete EC* with RTF1 1–486. c, Complete EC* with RTF1 1–554. d, Complete EC* with RTF1 1–586. e, Complete EC* with RTF1 486–710. f, Complete EC* with RTF1 571–710. g, Complete EC* with RTF1 587–710. h, Complete EC* with RTF1 601–710. i, RNA extension assays performed with RTF1 truncations. Quantification for fully extended product is shown for representative gel images found in A-H. Error bars represent the mean ± s.d. between three experimental replicates. j, RNA extension assays performed with RTF-6A mutant. Quantification for fully extended product is shown for representative gel images found in Fig. 5c. Error bars represent the mean ± s.d. between three experimental replicates. k, EC* readily forms a complex with RTF1 variant RTF1-6A. SDS-PAGE analysis of size exclusion chromatography fractions shows that RTF1-6A stably associates with EC*.

Extended Data Fig. 10 SPT5 and LEO1 truncations and their effects on RTF1 stimulation at 20 ºC.

a–k, RNA extension assays performed with SPT5 (residues 1–753) and PAF with LEO1 (residues 1–497) at 20 °C. Experiments were performed at least 3 times. a, Pol II. b, Pol II, DSIF, PAF. c, Pol II, DSIF, PAF, RTF1 d, Pol II, DSIF ∆CTR, PAF, RTF1. e, Pol II, DSIF, PAF ∆LEO1 1–497, RTF1. f, Pol II, DSIF ∆CTR, PAF ∆LEO1 1–497, RTF1. g, EC*. h, Complete EC*. i, Complete EC* with DSIF ∆CTR. j, Complete EC* with PAF LEO1 1–497 k, Complete EC* with DSIF ∆CTR and PAF LEO1 1-497. l, Quantification of complete RNA extension time course from 3 independent experiments at 20 °C with SPT5 ∆CTR and ∆LEO1 C-terminal extension. The extended product was quantified (Methods) and error bars correspond to mean ± s.d. between the replicate experiments. m, The same experiments as in L except SPT6 was also included.

Supplementary information

Supplementary Information

Supplementary Note 1 and Supplementary Tables 1, 3 and 4.

Reporting Summary

Supplementary Table 2

Crosslinking mass spectrometry raw data. Complete list of all BS3 crosslinks detected in complete EC*.

Supplementary Video 1

Overview of complete EC* structure.

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Source Data Fig. 1

Unprocessed gel images Fig. 1.

Source Data Fig. 1

Statistical source data Fig. 1.

Source Data Fig. 4

Unprocessed gel images Fig. 4.

Source Data Fig. 4

Statistical source data Fig. 4.

Source Data Fig. 5

Unprocessed gel images Fig. 5.

Source Data Fig. 5

Statistical source data Fig. 5.

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Vos, S.M., Farnung, L., Linden, A. et al. Structure of complete Pol II–DSIF–PAF–SPT6 transcription complex reveals RTF1 allosteric activation. Nat Struct Mol Biol 27, 668–677 (2020). https://doi.org/10.1038/s41594-020-0437-1

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