Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH - PubMed (original) (raw)

. 2015 Sep 3;59(5):794-806.

doi: 10.1016/j.molcel.2015.07.016.

Peter Cimermancic 2, Shruthi Viswanath 2, Christopher C Ebmeier 3, Bong Kim 1, Marine Dehecq 4, Vishnu Raman 3, Charles H Greenberg 2, Riccardo Pellarin 2, Andrej Sali 2, Dylan J Taatjes 3, Steven Hahn 5, Jeff Ranish 6

Affiliations

Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH

Jie Luo et al. Mol Cell. 2015.

Abstract

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit.

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Figures

Figure 1

Figure 1. Purification and crosslinking maps of human and yeast TFIIH

A) Silver stained gel of purified human TFIIH. B) Map of the identified inter-protein (red lines) and intra-protein (blue lines) crosslinks for human TFIIH. C) Silver stained gel of purified yeast TFIIH. D) Inter- and intra-protein crosslink map for yeast TFIIH as in (B). Red and green dots indicate the positions of lysine residues. Red dots indicate that the lysine residue was identified in a crosslink.

Figure 2

Figure 2. Molecular architecture of human and yeast TFIIH

A) Localization density map of the human core TFIIH subunits (top) and the best scoring model (bottom). The EM density map of human TFIIH used in this study is shown in grey mesh. B) Domain decomposition of human core TFIIH. C) Localization density map of the yeast TFIIH subunits (top) and the best scoring model (bottom). The EM density map of yeast TFIIH used in this study is shown in grey mesh. D) Domain decomposition of yeast TFIIH. The human and yeast models are superposed on the XPD/Rad3 subunit. The different shapes between the two models are due to differences in the cryo-EM density maps and may not represent actual structural differences between the two species, but are possibly due to artifacts due to different sample preparations and data collection as well as processing.

Figure 3

Figure 3. Summary of CXMS results for human and yeast TFIIH

A) Map of the identified inter-protein crosslinks for human (red lines) and yeast TFIIH (blue lines). Aligned sequences of human (bottom) and yeast (top) subunits are shown. Known and predicted structural domains are indicated as well as the positions of conserved topological regions identified in this study (orange bars). B) Summary of identified domain-domain crosslinks for human and yeast TFIIH. The thickness of the line is proportional to the number of crosslinks identified for each domain-domain linkage. The number of crosslinks supporting each domain-domain linkage is provided.

Figure 4

Figure 4. Crosslinks involving p62/Tfb1, XPD/Rad3, p44/Ssl1, and p34/Tfb4

Identified inter- and intra-protein crosslinks for human (red lines) and yeast (blue lines) homologs are mapped onto their aligned sequences. Known and predicted structural domains are indicated as well as the position of the p62/Tfb1Anchor region (red bar).

Figure 5

Figure 5. The p62/Tfb1 Anchor region is essential for the structural integrity of TFIIH

A) Schematic of the Tfb1 serial deletions analyzed in this study along with results of growth and UV sensitivity assays. Red bar indicates lethal phenotype. B) Results of IP-western analyses of Tfb1 domain deletions. The results are normalized to IP levels in the wild type Tfb1 strain. Error bars represent SEM for n = 3. C) Co-immunoprecipitation analysis with flag-tagged WT p62 (lane 2) or Anchor region deletions 292–328 (lane 3) or 328–432 (lane 4) in HeLa cells. CTRL: untransfected cells (lane 1). Note human p62 residues 292–328 and 328–432 align to yeast Tfb1 residues 359–397 and 397–505, respectively.

Figure 6

Figure 6. Schematic model of TFIIH subunit organization highlighting conserved topological features

The conserved topological features identified in this study are underlined. Subunit coloring scheme is the same as in Figure 2.

Figure 7

Figure 7. Mapping disease-associated mutations in XPD onto the human TFIIH models

Average placement of XPD without its C-terminal domain (precision of 8.5 Å) in the human TFIIH models is shown on the left. Disease-associated mutations in XPD are shown as red spheres. The location of the subunits in the context of core TFIIH is indicated by the grey rectangle on the right.

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