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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Roeder, R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 (1996)
Kornberg, R. D. Mediator and the mechanism of transcriptional activation. Trends Biochem. Sci. 30, 235–239 (2005)
Takagi, Y. & Kornberg, R. D. Mediator as a general transcription factor. J. Biol. Chem. 281, 80–89 (2006)
Plaschka, C. et al. Architecture of the RNA polymerase II–Mediator core initiation complex. Nature 518, 376–380 (2015)
Nozawa, K., Schneider, T. R. & Cramer, P. Core Mediator structure at 3.4 Å extends model of transcription initiation complex. Nature 545, 248–251 (2017)
Plaschka, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016)
He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016)
He, Y., Fang, J., Taatjes, D. J. & Nogales, E. Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013)
Murakami, K. et al. Structure of an RNA polymerase II preinitiation complex. Proc. Natl Acad. Sci. USA 112, 13543–13548 (2015)
Robinson, P. J. et al. Structure of a complete Mediator–RNA polymerase II pre-initiation complex. Cell 166, 1411–1422 (2016)
Gibbons, B. J. et al. Subunit architecture of general transcription factor TFIIH. Proc. Natl Acad. Sci. USA 109, 1949–1954 (2012)
Schultz, P. et al. Molecular structure of human TFIIH. Cell 102, 599–607 (2000)
Chang, W. H. & Kornberg, R. D. Electron crystal structure of the transcription factor and DNA repair complex, core TFIIH. Cell 102, 609–613 (2000)
Compe, E. & Egly, J. M. TFIIH: when transcription met DNA repair. Nat. Rev. Mol. Cell Biol. 13, 343–354 (2012)
Svejstrup, J. Q. et al. Different forms of TFIIH for transcription and DNA repair: holo-TFIIH and a nucleotide excision repairosome. Cell 80, 21–28 (1995)
Guzder, S. N., Sung, P., Bailly, V., Prakash, L. & Prakash, S. RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature 369, 578–581 (1994)
Goodrich, J. A. & Tjian, R. Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. Cell 77, 145–156 (1994)
Moreland, R. J. et al. A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II. J. Biol. Chem. 274, 22127–22130 (1999)
Alekseev, S. et al. Transcription without XPB establishes a unified helicase-independent mechanism of promoter opening in eukaryotic gene expression. Mol. Cell 65, 504–514 (2017)
Feaver, W. J., Svejstrup, J. Q., Henry, N. L. & Kornberg, R. D. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell 79, 1103–1109 (1994)
Kim, Y. J., Björklund, S., Li, Y., Sayre, M. H. & Kornberg, R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77, 599–608 (1994)
Wong, K. H., Jin, Y. & Struhl, K. TFIIH phosphorylation of the Pol II CTD stimulates mediator dissociation from the preinitiation complex and promoter escape. Mol. Cell 54, 601–612 (2014)
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)
Luo, J. et al. Architecture of the human and yeast general transcription and DNA repair factor TFIIH. Mol. Cell 59, 794–806 (2015)
Murakami, K. et al. Architecture of an RNA polymerase II transcription pre-initiation complex. Science 342, 1238724 (2013)
Fishburn, J., Tomko, E., Galburt, E. & Hahn, S. Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation. Proc. Natl Acad. Sci. USA 112, 3961–3966 (2015)
Tirode, F., Busso, D., Coin, F. & Egly, J. M. Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7. Mol. Cell 3, 87–95 (1999)
Kainov, D. E., Vitorino, M., Cavarelli, J., Poterszman, A. & Egly, J.-M. Structural basis for group A trichothiodystrophy. Nat. Struct. Mol. Biol. 15, 980–984 (2008)
Wolski, S. C. et al. Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD. PLoS Biol. 6, e149 (2008)
Constantinescu-Aruxandei, D., Petrovic-Stojanovska, B., Penedo, J. C., White, M. F. & Naismith, J. H. Mechanism of DNA loading by the DNA repair helicase XPD. Nucleic Acids Res. 44, 2806–2815 (2016)
Kuper, J., Wolski, S. C., Michels, G. & Kisker, C. Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation. EMBO J. 31, 494–502 (2012)
Doerks, T., Huber, S., Buchner, E. & Bork, P. BSD: a novel domain in transcription factors and synapse-associated proteins. Trends Biochem. Sci. 27, 168–170 (2002)
Warfield, L., Luo, J., Ranish, J. & Hahn, S. Function of conserved topological regions within the Saccharomyces cerevisiae basal transcription factor TFIIH. Mol. Cell. Biol. 36, 2464–2475 (2016)
Stefanini, M., Botta, E., Lanzafame, M. & Orioli, D. Trichothiodystrophy: from basic mechanisms to clinical implications. DNA Repair (Amst.) 9, 2–10 (2010)
Oh, K. S. et al. Phenotypic heterogeneity in the XPB DNA helicase gene (ERCC3): xeroderma pigmentosum without and with Cockayne syndrome. Hum. Mutat. 27, 1092–1103 (2006)
Rossignol, M., Kolb-Cheynel, I. & Egly, J. M. Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J. 16, 1628–1637 (1997)
Edwards, A. M., Kane, C. M., Young, R. A. & Kornberg, R. D. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J. Biol. Chem. 266, 71–75 (1991)
Serizawa, H., Conaway, J. W. & Conaway, R. C. Phosphorylation of C-terminal domain of RNA polymerase II is not required in basal transcription. Nature 363, 371–374 (1993)
Maxon, M. E., Goodrich, J. A. & Tjian, R. Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance. Genes Dev. 8, 515–524 (1994)
Kim, T. K., Ebright, R. H. & Reinberg, D. Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1421 (2000)
Grünberg, S., Warfield, L. & Hahn, S. Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat. Struct. Mol. Biol. 19, 788–796 (2012)
Farnung, L., Vos, S. M., Wigge, C. & Cramer, P. Nucleosome–Chd1 structure and implications for chromatin remodelling. Nature 550, 539–542 (2017)
Schaeffer, L. et al. The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor. EMBO J. 13, 2388–2392 (1994)
Lin, Y. C., Choi, W. S. & Gralla, J. D. TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape. Nat. Struct. Mol. Biol. 12, 603–607 (2005)
Hwang, J. R. et al. A 3′ → 5′ XPB helicase defect in repair/transcription factor TFIIH of xeroderma pigmentosum group B affects both DNA repair and transcription. J. Biol. Chem. 271, 15898–15904 (1996)
Ohkuma, Y. & Roeder, R. G. Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation. Nature 368, 160–163 (1994)
Wigley, D. B. & Bowman, G. D. A glimpse into chromatin remodeling. Nat. Struct. Mol. Biol. 24, 498–500 (2017)
Larivière, L. et al. Structure of the Mediator head module. Nature 492, 448–451 (2012)
Tsai, K. L. et al. Mediator structure and rearrangements required for holoenzyme formation. Nature 544, 196–201 (2017)
Cramer, P., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863–1876 (2001)
Kostrewa, D. et al. RNA polymerase II–TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009)
Louder, R. K. et al. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly. Nature 531, 604–609 (2016)
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017)
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016)
Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016)
Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)
Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)
Suhre, K. & Sanejouand, Y.-H. ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res. 32, W610–4 (2004)
Matthies, H. & Strang, G. The solution of nonlinear finite element equations. Int. J. Numer. Methods Eng. 14, 1613–1626 (1979)
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)
Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protocols 5, 725–738 (2010)
Yang, J. et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12, 7–8 (2015)
Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014)
Bordoli, L. et al. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protocols 4, 1–13 (2009)
Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013)
Raman, S. et al. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins 77 (Suppl. 9), 89–99 (2009)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
Sainsbury, S., Niesser, J. & Cramer, P. Structure and function of the initially transcribing RNA polymerase II–TFIIB complex. Nature 493, 437–440 (2013)
Miwa, K. et al. Crystal structure of human general transcription factor TFIIE at atomic resolution. J. Mol. Biol. 428, 4258–4266 (2016)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Ye, J., Kandegedara, A., Martin, P. & Rosen, B. P. Crystal structure of the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-responsive repressor. J. Bacteriol. 187, 4214–4221 (2005)
Fadden, A. J. et al. A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes. Nucleic Acids Res. 41, 9741–9752 (2013)
Ahmad, M. U. D. et al. Structural insights into nonspecific binding of DNA by TrmBL2, an archaeal chromatin protein. J. Mol. Biol. 427, 3216–3229 (2015)
Gervais, V. et al. Solution structure of the N-terminal domain of the human TFIIH MAT1 subunit: new insights into the RING finger family. J. Biol. Chem. 276, 7457–7464 (2001)
Schmitt, D. R., Kuper, J., Elias, A. & Kisker, C. The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold. PLoS One 9, e102389 (2014)
Tempel, W. et al. Structural genomics of Pyrococcus furiosus: X-ray crystallography reveals 3D domain swapping in rubrerythrin. Proteins 57, 878–882 (2004)
Kellenberger, E. et al. Solution structure of the C-terminal domain of TFIIH P44 subunit reveals a novel type of C4C4 ring domain involved in protein-protein interactions. J. Biol. Chem. 280, 20785–20792 (2005)
Xu, D. & Zhang, Y. Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field. Proteins 80, 1715–1735 (2012)
Di Lello, P. et al. NMR structure of the amino-terminal domain from the Tfb1 subunit of TFIIH and characterization of its phosphoinositide and VP16 binding sites. Biochemistry 44, 7678–7686 (2005)
Kim, J. S. et al. Crystal structure of the Rad3/XPD regulatory domain of Ssl1/p44. J. Biol. Chem. 290, 8321–8330 (2015)
Fan, L. et al. Conserved XPB core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol. Cell 22, 27–37 (2006)
Hilario, E., Li, Y., Nobumori, Y., Liu, X. & Fan, L. Structure of the C-terminal half of human XPB helicase and the impact of the disease-causing mutation XP11BE. Acta Crystallogr. D 69, 237–246 (2013)
Baker, M. L. et al. Modeling protein structure at near atomic resolutions with Gorgon. J. Struct. Biol. 174, 360–373 (2011)
Baker, M. L., Baker, M. R., Hryc, C. F., Ju, T. & Chiu, W. Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps. Biopolymers 97, 655–668 (2012)
Okuda, M. et al. Structural insight into the TFIIE-TFIIH interaction: TFIIE and p53 share the binding region on TFIIH. EMBO J. 27, 1161–1171 (2008)
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996)
Trabuco, L. G., Villa, E., Mitra, K., Frank, J. & Schulten, K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673–683 (2008)
Jakhanwal, S., Lee, C. T., Urlaub, H. & Jahn, R. An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly. EMBO J. 36, 1788–1802 (2017)
Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012)
Combe, C. W., Fischer, L. & Rappsilber, J. xiNET: cross-link network maps with residue resolution. Mol. Cell. Proteomics 14, 1137–1147 (2015)
Kosinski, J. et al. Xlink Analyzer: software for analysis and visualization of cross-linking data in the context of three-dimensional structures. J. Struct. Biol. 189, 177–183 (2015)
Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010)
Fairman-Williams, M. E., Guenther, U.-P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010)
Gu, M. & Rice, C. M. Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism. Proc. Natl Acad. Sci. USA 107, 521–528 (2010)
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.
The authors declare no competing financial interests.
Reviewer Information Nature thanks S. Hahn, X. Zhang and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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.
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.
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.
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). f–k, 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.
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.
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.
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.
This file contains Supplementary Tables 1-4. (PDF 417 kb)
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)
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)
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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). https://doi.org/10.1038/nature24282
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