Structures of transcription pre-initiation complex with TFIIH and Mediator


For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC–core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the ‘E-bridge’ helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC–core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

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Figure 1: Structure of the Pol II PIC.
Figure 2: Structure of the PIC–cMed complex.
Figure 3: Structure of TFIIH.
Figure 4: Interactions of TFIIH with cPIC.
Figure 5: TFIIH and DNA opening.
Figure 6: TFIIH and phosphorylation of Pol II.

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We thank S. Neyer, C. Bernecky, C. Burzinski, S. Vos, L. Farnung and other members of the Cramer laboratory for help. We thank C.-T. Lee and I. Parfentev from the Urlaub group for mass spectrometry. H.U. was supported by the Deutsche Forschungsgemeinschaft (SFB860). P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935), the Advanced Grant TRANSREGULON (grant agreement no. 693023) of the European Research Council, and the Volkswagen Foundation.

Author information




S.S. carried out all experiments and data analysis except for the following. M.H. carried out EDC crosslinking and Mediator modelling. C.D. performed TFIIE modelling and established a protocol for cPIC formation. D.T. wrote and applied the WarpCraft software. C.W. supervised EM data collection. H.U. conducted mass spectrometry. P.C. designed and supervised research. S.S. and P.C. prepared the manuscript.

Corresponding author

Correspondence to P. Cramer.

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Reviewer Information Nature thanks S. Hahn, X. Zhang and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Preparation of TFIIH and the PIC–cMed complex.

a, Preparation of recombinant TFIIH. Analysis of purified TFIIH core and kinase modules by size-exclusion chromatography and SDS–PAGE revealed high purity and homogeneity of the complexes with apparently stoichiometric subunits. SDS–PAGE analysis of fractions 1–13 of a sucrose gradient centrifugation after reconstitution of TFIIH from purified core and kinase modules. A shift in the bands originating from the subunits of the kinase module (Ccl1, Kin28 and Tfb3) by four fractions was detected, indicating formation of complete TFIIH. This experiment was repeated four times with equivalent results. b, Assembly of complexes. SDS–PAGE analysis of fractions 1–19 of 15–40% sucrose gradient centrifugations (Methods). Labelling of protein subunits according to the colour scheme in Figs 1 and 2. The analysis demonstrates successful formation of the cPIC, cPIC–cMed and PIC–cMed complexes (top to bottom). Bands originating from Pol II, cMed and TFIIH are shifted by several fractions, indicating formation of higher-order complexes. Subunits are present in apparently stoichiometric amounts. This experiment was repeated three times with equivalent results. c, Representative cryo-EM micrograph of the PIC–cMed complex. A scale bar is provided. d, 2D-class averages reveal 2D reconstructions from particles with clear signal for TFIIH and/or cMed adjacent to the centrally located Pol II density. A scale bar is provided.

Extended Data Figure 2 Cryo-EM data processing and quality of reconstructions.

a, Particle sorting and classification tree used for 3D reconstruction of the PIC and PIC–cMed complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The distinct branches of the classification tree (Methods) are highlighted in pink (PIC) and blue (PIC–cMed). In a conventional focused refinement approach in RELION23,55, the best-resolved PIC class was reconstructed with a local TFIIH mask, resulting in a focused map with a nominal resolution of 7.4 Å (green branch) that was not deposited. b, Two views of the final reconstructions of PIC and PIC–cMed coloured according to local resolution6. The colour scheme is indicated. c, Fourier shell correlation (FSC) between half maps of the final reconstructions of PIC and PIC–cMed. Resolutions for the gold-standard FSC 0.143 criterion are listed. For comparison of distinct regions within PIC and PIC–cMed reconstructions, FSC 0.143 was additionally calculated using local masks. d. Angular distribution plot for all particles in the final reconstructions of PIC and PIC–cMed. Colour shading from blue to yellow correlates with the number of particles at a specific orientation as indicated.

Extended Data Figure 3 EDC crosslinking analysis of PIC–cMed.

a, EDC-derived inter-subunit crosslinks between selected subunits in the PIC–cMed complex. Observed crosslinks are consistent with the structure of the cPIC and with positions of previously reported BS3- and SBAT-crosslinks. Colour code as indicated. b, EDC-crosslinks observed in TFIIH and between TFIIH and cPIC. Intra- and inter-subunit crosslinks are depicted as blue and black lines, respectively. Crosslinks between the TFIIE Tfa1 C-terminal region and Tfb1, Tfb2 and Ssl1 confirm interactions between TFIIE elements and TFIIH. c, Crosslinking hub of the Tfb1 N-terminal region. Ribbon representation of Tfb1 (residues 1–353, 369–394 and 544-639) and the surrounding domains of Rad3, Ssl1 and Tfb4. BS3-/SBAT- and EDC-derived crosslinks are depicted in red and black, respectively. The displayed crosslinks aided modelling of the Tfb1 PHD, BSD1, BSD2 and Rad3 anchor domains into the cryo-EM density. d, Statistical analysis of EDC-derived crosslinks. Most observed crosslinks are within a cutoff Cα distance of 16 Å. Cα distances of up to 21 Å may be attributed to flexibility of the involved residues and the coordinate error of the model. Some outliers with Cα distances of 22–30 Å were observed for the well-defined cPIC and Rad3 structures and may have originated from over-crosslinking of particles.

Extended Data Figure 4 TFIIH structure and quality of the cryo-EM density.

a, Schematic of TFIIH subunit and domain architecture with bound double-stranded DNA (dsDNA) using the top view. Flexible linkers are depicted as black lines. Prominent helices within the folds of the tethering subunit Tfb1 and in Tfb2 are highlighted. b, Top view of the TFIIH structure in cylindrical representation. Prominent domains are labelled. The DNA register with respect to the putative transcription start site +1 is indicated. c, Overall fit of PIC structure into final WarpCraft PIC reconstruction. Observed density for a few remaining regions that could be clearly assigned but were not modelled are highlighted as indicated in Supplementary Table 1. d, Fit of cPIC structure into final WarpCraft PIC reconstruction at a higher contour level than in c shows the high resolution of the map in this region. e, Fit of TFIIH model into final WarpCraft PIC reconstruction. EM map reveals secondary structure throughout. Observed density for regions that could be clearly assigned but were not modelled are highlighted (compare with Supplementary Table 1). fk, EM density (black mesh) for domains and subunits of TFIIH reveals secondary structure throughout. Loops and linkers were traced when continuous density between unambiguously placed models was observed. Depicted density is part of either the WarpCraft PIC reconstruction or a focused reconstruction with a local mask on TFIIH core unless indicated otherwise. l, Cryo-EM reconstruction of the PIC reveals side-chain density in well-ordered regions. Depicted are helical regions in the large Pol II subunit Rpb1. m, Fit of the PIC–cMed model into the final WarpCraft PIC–cMed reconstruction. Structures of cMed head and middle modules account for density within this region.

Extended Data Figure 5 Location of essential regions in TFIIH and sites mutated in disease.

a, TFIIH regions essential for cell viability in yeast. Mapping of TFIIH regions identified to be essential in S. cerevisiae by in vivo deletion analysis33 on the PIC structure revealed that they are generally forming well-ordered regions of the TFIIH core. Structures are viewed from the top (Fig. 1) with regions coloured in magenta or yellow if their removal caused cell lethality or growth defects, respectively. Affected TFIIH subunits and ranges of deleted residues are highlighted in colours according to Fig. 3. For deletions exceeding the modelled residue range, the last modelled residue is indicated in parentheses. b, Mapping of human disease mutations onto the structures of Rad3 (human XPD) and Tfb5 (human p8). Reported mutations in xeroderma pigmentosum, trichothiodystrophy or Cockayne syndrome14,34,35 were included. The sites of point mutations are depicted as red spheres, and Tfb5 truncations are coloured in black. Colour coding of TFIIH subunits as in Fig. 3. A list of yeast residues highlighted in the PIC structure is provided together with the corresponding human mutations in parentheses. Mutation sites are conserved. Rad3 mutations apparently interfere either with the stability and/or the function of the ATPase core or with the Rad3–Ssl1 interaction. Only few mutations target the FeS cluster or ARCH domain. Newly available data on the Rad3 anchor in Tfb1 suggest close proximity to at least four mutation sites that may affect the Rad3–Tfb1 interaction in this region. Tfb5 mutations either abolish Ssl2 binding or the formation of the dimerization domain with the Tfb2 C terminus, resulting in destabilization of the Ssl2/Tfb2 region. If the clutch domains remain intact, however, a complete disruption of the Ssl2/Tfb2 interaction seems unlikely. We omitted Ssl2 from analysis as our structure does not cover the region in which reported mutations occur.

Extended Data Figure 6 TFIIE–TFIIH interactions.

a, Tfb3–Pol II interaction. The TFIIH kinase module subunit Tfb3 (human MAT1) tethers Pol II and the TFIIH core together. Ribbon representation of the Tfb3 N-terminal RING-finger binding in a groove between the Pol II stalk subunit Rpb7 and the TFIIE E-linker helices. The RING-finger is linked to the ARCH anchor which binds the ARCH domain of Rad3. b, Secondary structure and conservation of TFIIE subunit Tfa1 as determined with CONSURF92. Regions observed in the PIC and PIC–cMed structures are exceptionally well conserved throughout evolution. C-terminal residues with used crosslinks are indicated. c, E-dock. The predicted Tfa1 helix α7 is wedged between the TFIIE extended winged helix (eWH) domain situated on the Pol II clamp and the PHD of Tfb1 in the TFIIH core. α7 was not modelled owing to weak density at the interface of the two major mobile parts of the PIC structure (cPIC and TFIIH) and owing to the absence of crosslinks (Methods). The Tfb1 PHD is additionally contacted by the Tfa1 C-terminal acidic region. The identity and directionality of this acidic peptide were unambiguously established by crosslinking (Methods). d, e, E-bridge. This helix (α8) extends from the Tfb1 BSD2 domain at the centre of the TFIIH crescent to the central β-sheet of the Ssl2 ATPase lobe 2. The C-terminal anchor peptide (dashed line) was not modelled into the density due to limited resolution. The identity and directionality of the E-bridge was unambiguously established by independent crosslinking experiments (Methods). f, g, E-floater. The Tfa1 helix α9 is positioned by the BSD1 domain of Tfb1 and located adjacent to the 3-helix bundle at the centre of the TFIIH crescent. The identity and directionality of the E-floater was unambiguously established by independent crosslinking experiments (Methods).

Extended Data Figure 7 Detailed analysis of Ssl2 ATPase conformation and implications for translocase activity.

a, Overview of PIC complex with highlighted Ssl2 (human XPB) ATPase lobes 1 and 2 (in pink and burgundy, respectively) and interacting domains of Tfb2, Tfb5 and Tfa1. b, Detailed view on Ssl2 positioned on dsDNA in the presumed pre-translocation state. The ATP analogue AMP-PNP was present in the buffer but was not observed in the active site of the Ssl2 ATPase, supporting the model that we trapped the structure in the pre-translocation state. Register of covered nucleotides with respect to the putative TSS +1 is indicated. Highlighted helicase motifs were identified and assigned as described93. Yellow coloured motifs are involved in the DNA interaction, purple motifs participate in NTP binding and hydrolysis, and green motifs are involved in coupling of ATP hydrolysis to DNA binding. Both lobes of the ATPase contact both nucleic acid strands. c, Chd1 and Ssl2 ATPases are closely related on a structural level and share the same fold. The presumed post-translocation state of Ssl2 was modelled by separate alignment of ATPase lobe 1 and 2 to the respective lobes in the structure of Chd1 bound to an ATP analogue (PDB code 5O9G); the presumed pre-translocation state was modelled vice versa using the Ssl2 structure as reference model. In both states the structures overlap to a high degree. d, The Ssl2–DNA arrangement observed in the PIC structure resembles that of 3′–5′-directed rather than 5′–3′-directed members of the SF2 family. Superposition of the Ssl2–dsDNA structure with models of the NS3 (PDB code 3KQK)94 and T. acidophilum (Tac) Rad3 (PDB code 5H8W)30 ATPase domains reveals a closer resemblance of Ssl2 to the 3′–5′-helicase NS3. Additionally, the bound single-stranded (ss) DNA fragment in the NS3 model aligned well to the dsDNA in the Ssl2 structure whereas the bound fragment in the TacRad3 structure was positioned differently and did not exhibit a minor groove twist as observed for NS3 and Ssl2 in the respective position. e, Superposition of structures of TacRad3 and ScRad3 ATPase domains indicates very high level of structural homology. ATPase lobes 1 and 2 were superimposed separately to account for the absence of bound DNA in the ScRad3 structure. f, Putative movement of E-bridge and the Tfb2–Tfb5 dimerization domain upon Ssl2 transition from the presumed pre- to the presumed post-translocation state (grey and colour, respectively). Upon movement of lobe 2, the E-bridge may undergo a rotation-translation movement towards Pol II and against its own trajectory onto the central β-ribbon of the Ssl2 ATPase lobe 2. The flexible Tfb2–Tfb5 dimerization domain would swing towards Pol II.

Extended Data Figure 8 Structure and conformational changes of cMed.

a, Schematic representation of cMed subunits. Regions contributing to submodules are coloured as in the S. pombe cMed crystal structure5. Solid and dashed black lines refer to protein regions that were modelled as atomic or backbone models, respectively. b, Ribbon model of cMed coloured by type of structural model used for interpreting the cryo-EM density. Regions with backbone models based on the S. pombe cMed structure5, regions with atomic models inclusive of the PDB code, and de novo modelled regions are indicated in grey, orange and blue, respectively. c, Repositioning of the cMed middle module upon PIC binding. The structures of unbound cMed (khaki, PDB code 5N9J) and PIC–cMed complex (blue, this study) were superimposed on the cMed head module. The positions of the cMed middle module domains hook, knob, connector, plank and beam apparently undergo conformational changes upon PIC binding, as indicated by arrows. This may cause or enlarge two observed openings at the head–middle interface. d, PIC–cMed interactions. Structure of the PIC–cMed complex in two views. The three previously identified interfaces4 between cPIC and cMed are indicated. In interface A, the Mediator movable jaw (light blue) contacts the Pol II Rpb3–Rpb11 heterodimer (red/yellow), the dock domain (beige) and the TFIIB β-ribbon (green). In interface B, the Mediator spine domain (green) contacts helix H* of the Pol II stalk subunit Rpb4 (blue) with its Med22 helix H1, and the Mediator arm domain (violet) contacts Rpb4 with its Med8 helices H1 and H2. In interface C, the Mediator plank domain (pink) contacts the Pol II foot region (cyan) with its Med9 helix H2. Two newly observed EDC-crosslinks between Med9 helix H2 and the Pol II foot domain are indicated by black spheres. e. Mediator head–middle module interfaces. In the unbound S. pombe cMed X-ray structure, four interfaces (I–IV) were observed between the head and middle modules5. Owing to stretching of the beam, interfaces I and II are altered in the PIC-bound cMed structure. In the new conformation, the Med4 C-terminal region in the Mediator knob is flexible and does not contact the spine region (interface III). Interface IV between the shoulder and hook domains is lost. Mediator domains are coloured as in a.

Extended Data Table 1 Components of the PIC–cMed complex
Extended Data Table 2 Cryo-EM data collection and model statistics for the PIC and the PIC–cMed complex structures

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-4. (PDF 417 kb)

Life Sciences Reporting Summary (PDF 72 kb)

Supplementary Data

WarpCraft source code. This zipped file contains the C# code used to perform the flexible refinement and a precompiled binary. (ZIP 36926 kb)


Video showing an overview of the WarpCraft cryo-EM map and ribbon model of the PIC structure. (MP4 26520 kb)

Overview of the WarpCraft cryo-EM map and ribbon model of the PIC structure

Video showing an overview of the WarpCraft cryo-EM map and ribbon model of the PIC structure. (MP4 26520 kb)


Video showing an overview of the WarpCraft cryo-EM map and ribbon model of the PIC-cMed structure. (MP4 26695 kb)

Overview of the WarpCraft cryo-EM map and ribbon model of the PIC-cMed structure

Video showing an overview of the WarpCraft cryo-EM map and ribbon model of the PIC-cMed structure. (MP4 26695 kb)


Conformational changes in the cMed middle module between the crystal structure of free cMed5 and the observed PIC-bound state. (MP4 3932 kb)

Conformational changes in the cMed middle module

Conformational changes in the cMed middle module between the crystal structure of free cMed5 and the observed PIC-bound state. (MP4 3932 kb)

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Schilbach, S., Hantsche, M., Tegunov, D. et al. Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature 551, 204–209 (2017).

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