ABSTRACT
The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB's ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB's ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.
Subject(s)
Adenosine Triphosphatases/chemistry , Chaetomium/chemistry , DNA/genetics , Fungal Proteins/chemistry , Protein Subunits/chemistry , Transcription Factor TFIIH/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites , Chaetomium/genetics , Chaetomium/metabolism , Cloning, Molecular , Crystallography, X-Ray , DNA/metabolism , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Transcription Factor TFIIH/genetics , Transcription Factor TFIIH/metabolism , Transcription, GeneticABSTRACT
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.
