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Transcription preinitiation complex structure and dynamics provide insight into genetic diseases

Abstract

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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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. The structural models of apo-TFIIH and holo-PIC have been deposited in the PDB with accession codes 6O9M and 6O9L, respectively.

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Acknowledgements

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.

Author information

I.I. directed the study. C.Y., T.D., Y.H., J.A.T., S.E.T. and I.I. contributed to the design of the study. C.Y. and T.D performed model building and molecular simulations of the models. C.Y. performed coordinate refinement. C.Y., T.D., S.E.T. and I.I. analyzed the data. C.Y., T.D., S.E.T., Y.H., J.A.T. and I.I. wrote the manuscript.

Correspondence to Ivaylo Ivanov.

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Integrated supplementary information

Supplementary Fig. 1 EM densities and corresponding atomic models for all TFIIH subunits.

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.

Supplementary Fig. 2 Positioning of the CAK subcomplex in the PIC.

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.

Supplementary Fig. 3 Mapping of CX-MS crosslinks onto the structure of TFIIH.

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.

Supplementary Fig. 4 Comparison of TFIIE–TFIIH binding interfaces for human and yeast PIC.

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.

Supplementary Fig. 5 Modeling TFIIEα-p62 interface of human holo-PIC.

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.

Supplementary Fig. 6 Mapping of human disease mutations onto the TFIIH subunits.

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 Information

Supplementary Information

Supplementary Figs, Supplementary Notes and Supplementary Tables

Reporting Summary

Supplementary Video 1

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.

Supplementary Video 2

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.

Supplementary Video 3

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.

Supplementary Video 4

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.

Supplementary Video 5

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.

Supplementary Video 6

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).

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Fig. 1: TFIIH integrative structure model based on comparative analysis of cryo-EM densities reveals ‘molecular rigging’ formed by p62 and p44.
Fig. 2: TFIIE links human PIC to TFIIH.
Fig. 3: TFIIE, MAT1 and p62 are critical for the integrity of the the core-PIC–TFIIH interface.
Fig. 4: Community networks underlying TFIIH functional dynamics.
Fig. 5: Pol II induces DNA bending and distortion near the initiation site.
Fig. 6: Human disease mutations mapped onto TFIIH and TFIIE show distinct patterns within protein–protein and community interfaces.
Supplementary Fig. 1: EM densities and corresponding atomic models for all TFIIH subunits.
Supplementary Fig. 2: Positioning of the CAK subcomplex in the PIC.
Supplementary Fig. 3: Mapping of CX-MS crosslinks onto the structure of TFIIH.
Supplementary Fig. 4: Comparison of TFIIE–TFIIH binding interfaces for human and yeast PIC.
Supplementary Fig. 5: Modeling TFIIEα-p62 interface of human holo-PIC.
Supplementary Fig. 6: Mapping of human disease mutations onto the TFIIH subunits.