Architecture of the RNA polymerase II–Mediator core initiation complex (original) (raw)

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Primary accessions

ArrayExpress

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

3D cryo-EM density maps of Pol II–DNA/RNA, cITC, and cITC–cMed have been deposited in the Electron Microscopy Database under accession numbers EMD-2784, EMD-2785, and EMD-2786, respectively. Coordinate files of Pol II–DNA/RNA, cITC, and cITC–cMed models have been deposited in the Protein Data Bank under accession numbers 4V1M, 4V1N, and 4V1O. Microarray data were deposited in ArrayExpress under accession number E-MTAB-2942.

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Acknowledgements

We thank C. Bernecky, W. Mühlbacher, B. Schwalb, P. Eser, S. Etzold from the Cramer laboratory and J. Plitzko and O. Mihalache from the Baumeister laboratory for help. We thank J. Schuller, S. Pfeffer and F. Förster for help with negative-stain tomography data acquisition and reconstruction of the initial reference. We thank N. Sessler and K. Makepace from the Borchers laboratory for help with mass spectrometry. We thank M. Raabe and H. Urlaub for protein identification. We thank S. Hahn for providing Srb5 (3× Flag) and TFIIH (TAP–Rad3) yeast strains. M.S. was supported by a Boehringer Ingelheim fellowship and the Elite Network of Bavaria. C.H.B. was supported by Genome Canada and Genome British Columbia Science and Technology Innovation Centre funding and The Natural Sciences and Engineering Research Council of Canada grant. F.H. was supported by grants of the LMU_excellent_ initiative, the Bavarian Research Center of Molecular Biosystems and the Deutsche Forschungsgemeinschaft/GRK1721. P.C. was supported by the Deutsche Forschungsgemeinschaft grants SFB646 and GRK1721 (the latter to P.C. and E.V.), the European Research Council Advanced Grant TRANSIT, the Jung-Stiftung, and the Volkswagen Foundation.

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany,
    C. Plaschka & P. Cramer
  2. Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany,
    L. Larivière, L. Wenzeck, M. Seizl, M. Hemann & F. Herzog
  3. Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany,
    D. Tegunov, W. Baumeister & E. Villa
  4. Department of Biochemistry and Microbiology, Genome British Columbia Protein Centre, University of Victoria, 3101-4464 Markham Street, Victoria, British Columbia V8Z7X8, Canada,
    E. V. Petrotchenko & C. H. Borchers
  5. Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA,
    E. Villa

Authors

  1. C. Plaschka
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  2. L. Larivière
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  3. L. Wenzeck
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  4. M. Seizl
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  5. M. Hemann
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  6. D. Tegunov
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  7. E. V. Petrotchenko
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  8. C. H. Borchers
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  9. W. Baumeister
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  10. F. Herzog
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  11. E. Villa
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  12. P. Cramer
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Contributions

C.P. designed and carried out all experiments, except for the following. L.L. performed initial cloning and purification of cMed and prepared the S. cerevisae head module homology model. L.W. assisted with protein purification and functional assays. M.S. carried out immobilized template and cDTA assays. M.H. carried out mass spectrometry of crosslinked cITC–cMed peptides. F.H. supervised mass spectrometry of cITC–cMed. E.V.P. and C.H.B. supervised mass spectrometry of cMed. W.B. provided essential materials and expertise. E.V. helped with EM data collection and supervised electron microscopy. E.V. and D.T. helped with EM data processing. P.C. designed and supervised research. C.P. and P.C. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence toP. Cramer.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 cMed reconstitution, activities, and cITC binding.

a, Cartoon view of the different reported positions of Mediator on Pol II. Yeast Mediator density was observed at four different locations on Pol II (orange left19, middle13,85,86, right15, green22). Another Mediator position was obtained in the human Pol II–TFIIF–VP16–Mediator complex21 (purple). The Mediator position presented in this work is shown for comparison (blue). Pol II is oriented the same way in all views, approximately viewed from the back. b, Schematic view of vectors used for co-expression of cMed. RBS, ribosome binding site; 10×His tag, 10×histidine tag; ori, origin of replication; lacI, gene encoding Lac repressor. Sm, Amp and Kan refer to streptomycin, ampicillin and kanamycin resistance genes, respectively. c, Recombinant cMed is active in activator-independent promoter-dependent transcription53. Compare with Fig. 1b. d, Recombinant cMed binds purified GST–CTD fusion protein, but not GST alone that was immobilized on glutathione resin. An asterisk marks two contaminant bands. e, Recombinant cMed binds Pol II in a pull-down experiment. Pol II was immobilized on streptavidin resin via a biotin tag on the Rpb3 subunit. f, Recombinant cMed binds purified Pol II during size exclusion chromatography. Chromatograms of cMed (blue), Pol II (grey) and Pol II–cMed complex (magenta) are shown (top). Peak fractions of the Pol II–cMed were analysed by SDS–PAGE (bottom). g, Negative-stain EM image of cITC–cMed complex. The scale bar is 100 nm. h, Cryo-EM image of cITC–cMed complex.

Extended Data Figure 2 Structural modelling of cITC into the cryo-EM density.

a, Automatic fitting of structures into cITC cryo-EM density. The order of structure fitting and the corresponding translation correlation peaks are indicated (left). After fitting of all structures (right), the remaining density (cyan) was attributed mainly to DNA and TFIIF. b, Fit of TBP to the cITC EM density. c, Detailed views of Pol II domains foot, funnel, and clamp head in the cITC EM density. d, Two views of the cITC EM reconstruction corresponding to previously defined front and side views of Pol II47. The final cITC model is coloured (DNA template/non-template, blue/cyan; RNA, red; Pol II, silver; TBP, red; TFIIB, green; TFIIF Tfg1/Tfg2, violet/magenta). Tfg1 and Tfg2 contain non-conserved insertions in the TFIIF dimerization module. e, The cITC map reveals density for the Tfg2 C-terminal linker and winged helix (WH) domain. The view corresponds to the side view in c, but is rotated around a vertical axis by 30°. The mobile Tfg2 WH domain is visible at lower density threshold. The homologous human RAP30 WH domain (Homo sapiens, Hs) was modelled on the basis of its position in the human initiation complex7 and resides at a similar location. f, The cITC map reveals density that may correspond to the Tfg1 N terminus, as suggested by protein–protein crosslinking9,30. g, Comparison of EM densities for promoter DNA in cITC (top) and in the Pol II–DNA/RNA complex (bottom). The Pol II elements active site (magenta), bridge helix (green), trigger loop (dark red), wall (grey), and template DNA (dark blue), non-template DNA (light blue) and RNA (red) are depicted. h, Minor repositioning of TFIIB cyclin domains compared to our previous model9. i, Minor rotation of the TBP–DNA–TFIIB complex on the Pol II wall in the cITC structure compared to the previous open complex model9.

Extended Data Figure 3 Structural modelling of cITC–cMed into the cryo-EM density.

a, Automatic fitting of atomic models into the cITC–cMed density. The order of model fitting and the corresponding translation correlation peaks are indicated (left). After fitting of all models (right), the remaining density (cyan) was attributed to the middle module and to some minor additional protein regions in the cITC and head module. b, Detailed views of Pol II domains foot, funnel, and clamp head in the cITC–cMed EM density. c, Fit of improved Mediator head crystallographic model to the corresponding cryo-EM density for cITC–cMed. The different head domains are depicted in different colours. Views are from the previously defined right side and top views of the head module14. d, The head module undergoes minor changes in conformation upon formation of the cITC–cMed complex. The EM fit with a modified model of the S. cerevisiae (Sc) Mediator head is compared to the crystal structures of head modules from S. pombe (Sp) (PDB code 4H63) and Sc (modified based on PDB code 4GWP). Previously defined top view of the head module14.

Extended Data Figure 4 Protein–protein crosslinking.

a, Crosslinks between Pol II and TBP, TFIIB, and TFIIF confirm cITC architecture. Crosslinks were visualized using xiNET (Rappsilber laboratory). b, Crosslinks within the cMed head module and between the head module and Pol II. Solid lines indicate crosslinks derived from cITC–cMed data; dashed lines indicate additional crosslinks obtained only for free cMed. c, Crosslinks within the cMed middle module and between the middle module and Pol II. Crosslinks within the middle module agree well with the proposed middle module architecture25,87. d, Crosslinks between head and middle modules elucidate cMed architecture. e, Possible location of the previous model of the Mediator middle module (ribbons, left25) within the observed cMed density in the cITC–cMed reconstruction based on protein crosslinking. The cITC–cMed complex is viewed from the ‘bottom’, which corresponds to the top view rotated by 180° around a vertical axis.

Extended Data Figure 5 Analysis of the cITC–cMed interface.

a, Crosslinks between cITC and cMed were mapped on available models of cITC and Mediator head module. Two views of cITC–cMed are shown, a top view rotated by 135° around the horizontal axis and an additional 90° around the same axis. Mapped crosslinks exceeding the 30 Å distance restraint were coloured in grey. Crosslinks between lysine residues (black spheres) are labelled or connect (dashed magenta lines) to proximal cMed density, where plausible. Med14 N- and C-terminal regions are indicated. b, Immobilized template assay using nuclear extract of srb4-ts and wild-type yeast strains demonstrates that recruitment of Pol II and TFIIB depends on the Mediator head module. Schematic protocol for the immobilized template (ITA) assay53 (top). The loss of Pol II, TFIIB and Mediator head module from promoter DNA by heat inactivation of the srb4-ts nuclear extract is rescued by addition of recombinant head (rHead) module53 (bottom). c, Pull-down assays with recombinant cMed variants carrying mutations in interfaces A, B, and C reveal that interface B is essential for Pol II binding, whereas interfaces A and C are not. For definition of interfaces see Fig. 5. The assay measures retention of cMed variants on Pol II coupled to beads (see Extended Data Fig. 1e). Interface A was perturbed by deletion of the movable jaw, Med8C(residues 1–189)–Med18–Med20. Interface B was perturbed by removal of parts of the arm domain, either by N-terminal truncation of Med8 (Δ1–29, deleting only the flexible N-terminal tail; Δ1–59, deleting the tail and helix α1; Δ1–87, deleting the tail and helices α1 and α2), or by removal of Med6, which contributes a helix to the arm domain (ΔMed6). Interface C was perturbed by deletion of Med4–Med9. Shown are SDS–PAGE analyses (Coomassie staining). On the left, 2 μg of purified input cMed variants were analysed and the integrity of the complexes confirmed. The identity of the cMed variant-specific bands was confirmed by mass spectrometry. Some minor contaminant bands are marked with asterisks. On the middle gel, the bead elutions after the pull-down assays are shown. Wild-type cMed bound to Pol II-coupled beads, providing a positive control (rightmost lane). cMed variants were retained by Pol II-coupled beads to various degrees. Whereas cMed variants with perturbations in interfaces A and C bound to Pol II, several variants with perturbations in interface B were impaired for Pol II binding. On the right, the bead controls are shown. None of the analysed cMed variants bound to beads only, providing a negative control.

Extended Data Figure 6 Global requirement of the Mediator head module for transcription.

a, Fold changes in RNA degradation (log folds, _x_-axis) and synthesis (log folds, _y_-axis) rates observed in strains srb4-ts versus wild-type in the absence of heat shock. Each point corresponds to one mRNA and the density of points is reflected in their brightness. Red contour lines define regions of equal intensity. The centre of the distribution results from the median synthesis and degradation rates, whose relative contributions are indicated by shifts of the red lines parallel to synthesis or degradation rate axis, respectively. b, Global shutdown of RNA synthesis upon heat shock (HS) of the srb4-ts mutant. Fold changes in degradation (log fold, _x_-axis) and synthesis (log folds, _y_-axis) rates of srb4-ts and wild-type strains are indicated, after 18 or 60 min of HS treatment, respectively. c, As for a, but using a Med18-anchor-away (AA) strain in absence of rapamycin (rap) treatment. d, Global downregulation of RNA synthesis upon anchor-away of the Med18 subunit, after 18 or 60 min of rapamycin (rap) treatment, as in b.

Extended Data Figure 7 Inferred locations of the Mediator tail module, TFIIS, the general factors TFIIA, TFIIE, and TFIIH, and the CTD, and comparison with the human initiation complex.

a, Superposition of revised free yeast Mediator EM reconstruction17 (transparent lower-resolution surface, coloured in shades of blue) onto cITC–cMed (coloured as in Fig. 3). This reveals a high degree of similarity in the cMed region, and suggests locations of the tail module and subunit Med1 that are not present in cMed. b, Modelled location of TFIIS (yellow) within the cITC–cMed complex based on the Pol II–TFIIS complex (PDB code 1Y1V)48. c, Movement of the Rpb4–Rpb7 stalk upon cMed binding to the cITC and location of the ‘cradle’. Two views of the S. cerevisiae (Sc) cITC–cMed complex related by a 180° rotation around a vertical axis. The left view corresponds to the previously defined top view of Pol II47. The box contains a zoom-in view of the Rpb4–Rpb7 complex revealing its movement upon cMed binding. The right view reveals the location of the ‘cradle’ on the ‘bottom’ of Pol II. The density is transparent, with the final cITC-head module model underneath and coloured (DNA template/non-template, blue/cyan; RNA, red; Pol II, silver; TBP, red; TFIIB, green; TFIIF Tfg1/Tfg2, violet/magenta; head module, blue; middle module, violet). d, Human (Homo sapiens, Hs) initiation complex EM reconstruction7 viewed as in a. The complex reveals equivalent locations for TFIIB, TBP, TFIIF, and DNA, and additionally contains TFIIA (yellow), TFIIE (magenta), and the core subcomplex of TFIIH (solid orange surface) and a modelled TFIIH kinase subcomplex (orange mesh). e, Putative density for the CTD (magenta) near a modelled peptide (black) positioned according to the head module-CTD complex co-crystal structure (PDB code 4GWQ). The putative density superposes moderately well with the modelled CTD peptide and suggests that the CTD may adopt a different conformation in cITC–cMed with respect to the binary head module–CTD complex. This density cannot be assigned to the CTD with certainty because it may also stem from unresolved protein regions in Mediator. Coloured as in Fig. 5a. The region C-terminal of the CTD remained flexible and crosslinked to distant proteins (not shown, see Extended Data Fig. 4). f, Recombinant cMed stimulates TFIIH kinase activity, whereas the free head and middle modules do not. The relative activity (rel act) of Pol II phosphorylation at CTD residue serine-5 (S5P) was determined with respect to background (Rpb3).

Extended Data Figure 8 3D classification of negative-stain and cryo-EM data.

a, 3D classification of the negative-stain EM data set into four classes. The percentage of data in each class is given in parenthesis. To help visualize structural differences, all 3D reconstructions were radially coloured in UCSF Chimera. b, Pseudo-hierarchical 3D classification of the cryo-EM data set. The percentage of the data in each class is given in parentheses. Rejects refer to EM reconstructions that did not reflect the known structures of Pol II, cITC or cITC–cMed. §, class of partial cITC–cMed particles lacking upstream DNA–TFIIF–TFIIB–TBP; +, class of partial cITC–cMed particles lacking upstream DNA–TFIIB–TBP and the mobile plank of cMed; Fig. 5d. The Pol II–TFIIF class (Round 6) presented with density for the TFIIF dimerization module and the Tfg1 ‘charged helix’, but weak to no density for Tfg1 ‘arm’ and Tfg2 ‘linker’ regions due to the absence of upstream DNA stabilizing factors TFIIB and TBP. Classes were visualized as in a. c, 3D classification of particles from rejects of round 1 using the Pol II–DNA/RNA reconstruction as initial model. Particles were sorted into eight groups, resulting in poor 3D reconstructions. Classes were visualized as in a. d, 3D classification of particles from class 4 of round 1 using the Pol II–DNA/RNA reconstruction as initial model. Particles were sorted into four groups, resulting in EM reconstructions of cITC and cITC–cMed. These results suggest high data quality, and further the presence of a single detectable cITC–cMed conformation in the cryo-EM data, even in the absence of a cITC–cMed reference. Classes were visualized as in a.

Extended Data Figure 9 Negative stain and cryo-EM reconstructions of Pol II–DNA/RNA, cITC, and cITC–cMed complexes.

a, Four views of the negative-stain tomography reconstruction of the cITC–cMed related by 90° rotation, starting from the previously defined front view of Pol II47. b, Comparison of five reference-free 2D class averages calculated from all particles used in the final negative-stain single-particle reconstruction with corresponding forward projections of the reconstruction. c, Orientational distribution plot of all particles in the final negative-stain single-particle reconstruction. The estimated angular accuracy is 3.2°. d, Fourier shell correlation of the final negative-stain single-particle cITC–cMed reconstruction (0.143 FSC = 25.2 Å resolution bin). e, Four views of the negative-stain single-particle reconstruction of the cITC–cMed related by 90° rotation, starting from the previously defined front view of Pol II47. f, Comparison of five reference-free 2D class averages calculated from all particles used in the final Pol II–DNA/RNA cryo-EM single-particle reconstruction with corresponding forward projections of the reconstruction. g, Orientational distribution plot of all particles in the final cryo-EM Pol II–DNA/RNA single-particle reconstruction. The estimated angular accuracy is 3.2°. h, Fourier shell correlation of the final Pol II–DNA/RNA cryo-EM single-particle reconstruction (FSC = 0.143). i, Two views of the Pol II–DNA/RNA cryo-EM map are shown from the previously defined front view of Pol II47 and rotated by 180°, and are coloured by local resolution. j, Two views of the Pol II–DNA/RNA cryo-EM map are shown from the previously defined front view of Pol II47 and rotated by 180°, and are coloured by variance (the standard deviation, StdDev, of the normalized intensity value). k–o, As f–j but for the cITC reconstruction. p–t, As f–j but for the cITC–cMed reconstruction.

Extended Data Table 1 Components of the cITC–cMed complex

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Plaschka, C., Larivière, L., Wenzeck, L. et al. Architecture of the RNA polymerase II–Mediator core initiation complex.Nature 518, 376–380 (2015). https://doi.org/10.1038/nature14229

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