Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly’s global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE–p62 interactions that link core-PIC to TFIIH. p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations associated with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.
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Moore, M. J. & Proudfoot, N. J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688–700 (2009).
Proudfoot, N. J., Furger, A. & Dye, M. J. Integrating rnRNA processing with transcription. Cell 108, 501–512 (2002).
Bentley, D. L. Coupling mRNA processing with transcription in time and space. Nat. Rev. Genet. 15, 163–175 (2014).
Roeder, R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 (1996).
Zurita, M. & Cruz-Becerra, G. TFIIH: new discoveries regarding its mechanisms and impact on cancer treatment. J. Cancer 7, 2258–2265 (2016).
Hishikawa, A., Hayashi, K. & Itoh, H. Transcription factors as therapeutic targets in chronic kidney disease. Molecules 23, 1123–1136 (2018).
Villard, J. Transcription regulation and human diseases. Swiss Med. Wkly 134, 571–579 (2004).
Chen, X. F., Zhang, Y. W., Xu, H. X. & Bu, G. J. Transcriptional regulation and its misregulation in Alzheimer’s disease. Mol. Brain 6, 44–53 (2013).
Lee, T. I. & Young, R. A. Transcriptional regulation and its misregulation in disease. Cell 152, 1237–1251 (2013).
Goodrich, J. A., Cutler, G. & Tjian, R. Contacts in context: promoter specificity and macromolecular interactions in transcription. Cell 84, 825–830 (1996).
Boeger, H. et al. Structural basis of eukaryotic gene transcription. FEBS Lett. 579, 899–903 (2005).
Buratowski, S., Hahn, S., Guarente, L. & Sharp, P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549–561 (1989).
Liu, X., Bushnell, D. A., Silva, D. A., Huang, X. H. & Kornberg, R. D. Initiation complex structure and promoter proofreading. Science 333, 633–637 (2011).
Cheung, A. C. M., Sainsbury, S. & Cramer, P. Structural basis of initial RNA polymerase II transcription. Embo J. 30, 4755–4763 (2011).
Sim, R. J., Belotserkovskaya, R. & Reinberg, D. Elongation by RNA polymerase II: the short and long of it. Genes Dev. 18, 2437–2468 (2004).
He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016).
Schilbach, S. et al. Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature 551, 204–209 (2017).
He, Y., Fang, J., Taatjes, D. J. & Nogales, E. Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013).
Greber, B. J. et al. The cryo-electron microscopy structure of human transcription factor IIH. Nature 549, 414–417 (2017).
Dubaele, S. et al. Basal transcription defect discriminates between xeroderma pigmentosum and trichothiodystrophy in XPD patients. Mol. Cell 11, 1635–1646 (2003).
Coin, F. & Egly, J. M. Ten years of TFIIH. Cold Spring Harb. Symp. Quant. Biol. 63, 105–110 (1998).
Lehmann, A. R. The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases. Genes Dev. 15, 15–23 (2001).
Berneburg, M. & Lehmann, A. R. Xeroderma pigmentosum and related disorders: defects in DNA repair and transcription. Adv. Genet. 43, 71–102 (2001).
Fassihi, H. et al. Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect. Proc. Natl Acad. Sci. USA 113, E1236–E1245 (2016).
Boyle, J. et al. Persistence of repair proteins at unrepaired DNA damage distinguishes diseases with ERCC2 (XPD) mutations: cancer-prone xeroderma pigmentosum vs. non-cancer-prone trichothiodystrophy. Hum. Mutat. 29, 1194–1208 (2008).
Rimel, J. K. & Taatjes, D. J. The essential and multifunctional TFIIH complex. Protein Sci. 27, 1018–1037 (2018).
Compe, E. & Egly, J. M. Nucleotide excision repair and transcriptional regulation: TFIIH and Beyond. Annu. Rev. Biochem. 85, 265–290 (2016).
Singh, A., Compe, E., Le May, N. & Egly, J. M. TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription. Am. J. Hum. Genet. 96, 194–207 (2015).
Compe, E. & Egly, J. M. TFIIH: when transcription met DNA repair. Nat. Rev. Mol. Cell Biol. 13, 343–354 (2012).
Grunberg, S. & Hahn, S. Structural insights into transcription initiation by RNA polymerase II. Trends Biochem. Sci. 38, 603–611 (2013).
Grunberg, 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).
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).
Singharoy, A. et al. Molecular dynamics-based model refinement and validation for sub-5 angstrom cryo-electron microscopy maps. eLlife 5, e16105 (2016).
Luo, J. et al. Architecture of the human and yeast general transcription and DNA repair factor TFIIH. Mol. Cell 59, 794–806 (2015).
Zhu, Q. Z., Wani, G., Sharma, N. & Wani, A. Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair. DNA Repair 11, 942–950 (2012).
Drapkin, R., LeRoy, G., Cho, H., Akoulitchev, S. & Reinberg, D. Human cyclin-dependent kinase-activating kinase exists in three distinct complexes. Proc. Natl Acad. Sci. USA 93, 6488–6493 (1996).
Fuss, J. O. & Tainer, J. A. XPB and XPD helicases in TFIIH orchestrate DNA duplex opening and damage verification to coordinate repair with transcription and cell cycle via CAK kinase. DNA Repair 10, 697–713 (2011).
Coin, F., Oksenych, V. & Egly, J. M. Distinct roles for the XPB/p52 and XPD/p44 subcomplexes of TFIIH in damaged DNA opening during nucleotide excision repair. Mol. Cell 26, 245–256 (2007).
Fan, L. et al. XPD helicase structures and activities: Insights into the cancer and aging phenotypes from XPD mutations. Cell 133, 789–800 (2008).
Fan, L. & DuPrez, K. T. XPB: an unconventional SF2 DNA helicase. Prog. Biophys. Mol. Biol. 117, 174–181 (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).
Abdulrahman, W. et al. ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities. Proc. Natl Acad. Sci. USA 110, E633–E642 (2013).
Obmolova, G., Ban, C., Hsieh, P. & Yang, W. Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407, 703–710 (2000).
Mason, A. C. et al. A structure-specific nucleic acid-binding domain conserved among DNA repair proteins. Proc. Natl Acad. Sci. USA 111, 7618–7623 (2014).
Cheng, K. & Wigley, D. B. DNA translocation mechanism of an XPD family helicase. eLife 7, e42400 (2018).
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).
Ohkuma, Y., Hashimoto, S., Wang, C. K., Horikoshi, M. & Roeder, R. G. Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-alpha. Mol. Cell. Biol. 15, 4856–4866 (1995).
David, C. C. & Jacobs, D. J. Principal component analysis: a method for determining the essential dynamics of proteins. Methods Mol. Biol. 1084, 193–226 (2014).
Eargle, J. & Luthey-Schulten, Z. NetworkView: 3D display and analysis of protein center dot RNA interaction networks. Bioinformatics 28, 3000–3001 (2012).
Parvin, J. D., Shykind, B. M., Meyers, R. E., Kim, J. S. & Sharp, P. A. Multiple sets of basal factors initiate transcription by RNA-Polymerase-II. J. Biol. Chem. 269, 18414–18421 (1994).
Parvin, J. D. & Sharp, P. A. DNA topology and a minimal set of basal factors for transcription by RNA Polymerase-II. Cell 73, 533–540 (1993).
Ueda, T. et al. alleles contribute to the phenotype of compound heterozygote xeroderma pigmentosum patients. J. Exp. Med. 206, 3031–3046 (2009).
DiGiovanna, J. J. & Kraemer, K. H. Shining a light on xeroderma pigmentosum. J. Invest. Dermatol. 132, 785–796 (2012).
Kuschal, C. et al. GTF2E2 mutations destabilize the general transcription factor complex TFIIE in individuals with DNA repair-proficient trichothiodystrophy. Am. J. Hum. Genet. 98, 627–642 (2016).
Greber, B. J., Toso, D. B., Fang, J. & Nogales, E. The complete structure of the human TFIIH core complex. eLife 8, e44771 (2019).
Kurowski, M. A. & Bujnicki, J. M. GeneSilico protein structure prediction meta-server. Nucleic Acids Res. 31, 3305–3307 (2003).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Sali, A. & Blundell, T. L. Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).
Radu, L. et al. The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH. Nucleic Acids Res. 45, 10872–10883 (2017).
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).
Pettersen, E. F. et al. UCSF chimera - a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
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).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Case, D. A. et al. The Amber biomolecular simulation programs. J. Comput. Chem. 26, 1668–1688 (2005).
Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).
Tuckerman, M., Berne, B. J. & Martyna, G. J. Reversible multiple time scale molecular-dynamics. J. Chem. Phys. 97, 1990–2001 (1992).
Kale, L. et al. NAMD2: Greater scalability for parallel molecular dynamics. J. Comput. Phys. 151, 283–312 (1999).
Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).
Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).
Blanchet, C., Pasi, M., Zakrzewska, K. & Lavery, R. CURVES plus web server for analyzing and visualizing the helical, backbone and groove parameters of nucleic acid structures. Nucleic Acids Res. 39, W68–W73 (2011).
Amadei, A., Linssen, A. B. & Berendsen, H. J. Essential dynamics of proteins. Proteins 17, 412–425 (1993).
Roe, D. R. & Cheatham, T. E. 3rd PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095 (2013).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 27–28 (1996).
Glykos, N. M. Software news and updates. Carma: a molecular dynamics analysis program. J. Comput. Chem. 27, 1765–1768 (2006).
We thank S. Schilbach and P. Cramer for sharing the EMD-3846 cryo-EM density map and model before these became available from EMDB. We thank E. Nogales for insightful discussions. This work was supported by National Institutes of Health (NIH) grant No. GM110387 to I.I. Work on TFIIH by Y.H., S.E.T. and J.A.T. is supported by NIH grant No. P01 CA092584. J.A.T. is also supported for structural analyses by NIH grant No. R35 CA220430, a Robert A. Welch Chemistry Chair and the Cancer Prevention and Research Institute of Texas (Nos. RR140052 and RP180813). Computational resources were provided in part by an allocation from the National Science Foundation XSEDE program (No. CHE110042) to I.I. An award of computer time to I.I. was provided by the INCITE program. This research also used resources of the Oak Ridge Leadership Computing Facility, which is a DOE Office of Science User Facility supported under Contract No. DE-AC05-00OR22725.
The authors declare no competing interests.
Publisher’s note:Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a, Overall fit of the apo-TFIIH structure to the apo-TFIIH density (EMD-3802). The structural motifs were labeled for each TFIIH subunit. b-f, TFIIH subunits and domains flexibly fitted into the EM density. Domains in each subunit are indicated with red dashed circles. The yeast Tfb3 density is shown in purple in panel g. h, p52 and XPB. i, XPB and p44. j, p34 and p44. k, p62, p34 and p44. l, p62 and p34. m, p62 and XPD.
a,b, Two views of the human PIC with the CAK subcomplex highlighted. c,d, Extra cryoEM density in the holo-PIC model attributed to the CAK subcomplex (light blue) and to Rpb1 CTD (red), respectively. Gray surface show cryo-EM density EMD-3307. e, Schematic representation of CAK positioning in apo-TFIIH. Black dashed lines indicate the MAT1 linker between CAK and the 86-Å long helix of MAT1. f, Schematic representation of CAK positioning in our holo-PIC model. Pol II CTD domain is shown by a red dashed line. g, Schematic representation of yeast PIC-Mediator complex. Pol II CTD domain is shown by red dashed lines.
a, Intermolecular crosslinks in TFIIH. Crosslinks are color-coded as follows: black lines denote Cα–Cα distances <30 Å; green lines denote distances over 30 Å. b, Sequence alignment of human p62 (residues 1–162) to yeast Tfb1 (residues 1–229). c, The position of the p62 PHD domain relative to XPB and TFIIEα in human holo-PIC. Crosslinks between XPB and PHD are shown as colored spheres. d, Position of Tfb1 PHD domain relative to Ssl2 and Tfa1 in yeast.
a, TFIIEα-p62 interface in the human holo-PIC. Domains and secondary structure elements are labeled in TFIIEα and p62. XPD, XPB, p52, p44, p34, p8 and MAT1 are shown in surface representations. Core of PIC is shown in grey surface. TFIIEα (dark green) and p62 (blue) are displayed in cartoon representation. DNA is shown in cyan. b, Tfa1-Tfb1 interface in the yeast PIC. The position of the α7 helix is shown by a dashed red rectangle. Domains and secondary structure elements are labeled in Tfa1 and Tfb1. Tfa1 (green) and Tfb1 (blue) are displayed in cartoon representation. DNA is shown in cyan. Rad3, Ssl2, Tfb2, Ssl1, Tfb4 and Tfb4 are shown in surface representations. Core of PIC is shown in grey surface.
a, TFIIEα-p62 interface fitted in the holo-PIC density. DNA is shown in cyan. TFIIEα (Tfa1 in yeast) and p62 (Tfb1 in yeast) are shown in dark green and blue cartoon representation, respectively. Circles demark zoomed regions in panels b-e. b, The TFIIEα α7 helix is modeled from the holo-PIC density (EMD-3307). c, The TFIIEα α9 helix and p62 BSD1 domain are modeled from the yeast density (EMD-3846). d, Two views of TFIIEα acidic patch/α8 helix interaction and the p62 PHD domain in the holo-PIC density (EMD-3307). e, The TFIIEα β5 strand and the p62 BSD2 domain are modeled from the yeast density (EMD-3846). f, Conservation of TFIIEα.
a, Mapping of disease mutations (XP (black), TTD (green), XP/TTD (pink) and XP/CS (coral)) onto the XPD. The sites of point mutations are depicted as spheres. b, Mapping of disease mutations onto the XPB and p8. c, XPD mutations in the XPD-p62 interface are circled. d, XPD mutations in the XPD-p62 interface are circled. e, Engineered mutations in the p34-p44 and p44-XPD interfaces. f, Deletion analysis in TFIIH. Deletion of residues 328–432 of p62 has previously been found to prevent co-purification of XPD and XPB. XPB ATPase stimulation was found to depend on the second XPB-interacting domain in p52 (residues 305–462). The absence of a single subunit in p44 (deletion of residues 321–395) was found to abolish TFIIH transcription.
Supplementary Figs, Supplementary Notes and Supplementary Tables
Atomistic holo-PIC model fitted into the EM density of the closed complex PIC. PIC is shown in cartoon representation and colored as denoted in the video inset. The CC PIC cryo-EM density is shown in gray.
Positioning of the cyclin-activating kinase subcomplex within human holo-PIC. PIC is shown in cartoon representation and colored as denoted in the video inset. The CC PIC Cryo-EM density is shown in gray. The video zooms in on the CAK module and rotates the complex to provide in-depth view of the kinase module position.
Motion of the TFIIH communities along the second principal component PC2. PC2 shows the out-of-plane twisting movement of the TFIIH subunits with respect to Pol II core. TFIIH subunits are colored as dented in the video inset.
Motion of the TFIIH communities along the third principal component PC3. PC3 shows the in-plane swing motion of TFIIH that could push the DNA substrate toward the Pol II cleft. TFIIH subunits are colored as dented in the video inset.
Mapping of XP, XP/CS and TTD patient-derived mutations onto the TFIIH structure. XP/CS, XP, XP/TTD and TTD patient-derived mutations are mapped to TFIIH model. Point mutations are shown as spheres and colored by phenotype (see inset). Coloring of the TFIIH subunits is denoted in the video inset.
Motion of TFIIH communities along the PC3 mode with XP, XP/CS and TTD mutants mapped onto the structure. XP/CS, XP, XP/TTD and TTD patient-derived mutations are mapped to TFIIH model. The motion of the complex follows PC3. Point mutations are shown as spheres and colored by phenotype (coloring scheme follows video S5). Dynamic communities are shown (coloring scheme follows Figure 4).