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The complete structure of the human TFIIH core complex.

ABSTRACT: Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the architecture of TFIIH and its interactions within transcription pre-initiation complexes. Here, we present the complete structure of the human TFIIH core complex, determined by phase-plate cryo-electron microscopy at 3.7 Å resolution. The structure uncovers the molecular basis of TFIIH assembly, revealing how the recruitment of XPB by p52 depends on a pseudo-symmetric dimer of homologous domains in these two proteins. The structure also suggests a function for p62 in the regulation of XPD, and allows the mapping of previously unresolved human disease mutations.

SUBMITTER: Greber BJ

PROVIDER: S-EPMC6422496 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The complete structure of the human TFIIH core complex.

Greber Basil J BJ Toso Daniel B DB Fang Jie J Nogales Eva E

eLife 20190312

Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the archi ...[more]

PMID: 30860024

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Project description:Aims:Spironolactone (SPL) improves endothelial dysfunction and survival in heart failure. Immune modulation, including poorly understood mineralocorticoid receptor (MR)-independent effects of SPL might contribute to these benefits and possibly be useful in other inflammatory cardiovascular diseases such as pulmonary arterial hypertension. Methods and results:Using human embryonic kidney cells (HEK 293) expressing specific nuclear receptors, SPL suppressed NF-?B and AP-1 reporter activity independent of MR and other recognized nuclear receptor partners. NF-?B and AP-1 DNA binding were not affected by SPL and protein synthesis blockade did not interfere with SPL-induced suppression of inflammatory signalling. In contrast, proteasome blockade to inhibit degradation of xeroderma pigmentosum group B complementing protein (XPB), a subunit of the general transcription factor TFIIH, or XPB overexpression both prevented SPL-mediated suppression of inflammation. Similar to HEK 293 cells, a proteasome inhibitor blocked XPB loss and SPL suppression of AP-1 induced target genes in human pulmonary artery endothelial cells (PAECs). Unlike SPL, eplerenone (EPL) did not cause XPB degradation and failed to similarly suppress inflammatory signalling. SPL combined with siRNA XPB knockdown further reduced XPB protein levels and had the greatest effect on PAEC inflammatory gene transcription. Using chromatin-immunoprecipitation, PAEC target gene susceptibility to SPL was associated with low basal RNA polymerase II (RNAPII) occupancy and TNF?-induced RNAPII and XPB recruitment. XP patient-derived fibroblasts carrying an N-terminal but not C-terminal XPB mutations were insensitive to both SPL-mediated XPB degradation and TNF?-induced target gene suppression. Importantly, SPL treatment decreased whole lung XPB protein levels in a monocrotaline rat model of pulmonary hypertension and reduced inflammatory markers in an observational cohort of PAH patients. Conclusion:SPL has important anti-inflammatory effects independent of aldosterone and MR, not shared with EPL. Drug-induced, proteasome-dependent XPB degradation may be a useful therapeutic approach in cardiovascular diseases driven by inflammation.

Project description:Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2+E3BP) core organisation: the 'addition' model (60+12) and the 'substitution' model (48+12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the 'substitution' model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.

Dataset Information

The complete structure of the human TFIIH core complex.

Publications

The complete structure of the human TFIIH core complex.

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