The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH

Abstract

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein–protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.

Structural basis of TFIIH activation for nucleotide excision repair

“…The final map was denoised in Warp 1.0.6 46 . Structures of ATPase lobes 1 and 2 of XPB, XPD, p44 vWA-like domain, and p52 C-terminus (residues 383–458) from the TFIIH structure (PDB code 5OF4 [10.2210/pdb5OF4/pdb]) 6 , as well as the crystal structure of p34 vWA-like domain bound to p44 RING domain (PDB code 5O85 [10.2210/pdb5O85/pdb]) 50 were rigid-body fitted into our cryo-EM density in UCSF Chimera 49 and manually adjusted in COOT 51 . Owing to high quality of the EM density, the NTE domain and part of the DRD domain (residues 71–199 and 266–300), as well as the p52 region that interacts with XPB (residues 307–382) were built de novo guided by secondary structure prediction in PSIPRED 52 and bulky amino acid side chains as sequence registers.…”

Structural basis of TFIIH activation for nucleotide excision repair

“…The final map was denoised in Warp 1.0.6 48 . Structures of ATPase lobe 1 and 2 of XPB, XPD, p44 vWA-like domain and p52 C-terminus (residues 383-458) from the TFIIH structure (PDB code 5OF4) 6 , as well as the crystal structure of p34 vWA-like domain bound to p44 RING domain (PDB code 5O85) 51 were rigid-body fitted into our cryo-EM density in UCSF Chimera 35 and manually adjusted in COOT 52 . Due to high quality of the EM density, the NTE domain and part of the DRD domain (residues 71-199 and 266-300), as well as the p52 region that interacts with XPB (residues 290-382) were built de novo guided by secondary structure prediction in PSIPRED 53 and bulky amino acid side chains as sequence registers.…”

How to limit the speed of a motor: The intricate regulation of the XPB ATPase and Translocase in TFIIH

“…The modular composition of p52, however, may also be necessary to provide the flexibility to assume different conformational states to fulfill TFIIH's functions in transcription and NER (25)(26)(27)40). Importantly, the high structural homology between the C. thermophilum and the human proteins indicates a high functional homology of the two organisms that has been described and utilized previously (11,41). In this study, we extended the utilisation of this system towards the analysis of XPB regulation and the functional assembly of core TFIIH.…”