Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases

Abstract

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH’s global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Uncoupling the TFIIH Core and Kinase Modules Leads To Misregulated RNA Polymerase II CTD Serine 5 Phosphorylation

“…While many mutations in Core subunits therefore cause sensitivity to ultraviolet (UV) light, this is not the case for Kinase Module mutants. Recent structures (reviewed in (Yu et al, 2023)) show that the Kinase Module would block interactions between the Core Module and other NER factors. Therefore, TFIIH either enters into the NER complex as free Core Module, or the Kinase Module must dissociate soon after.…”

“…Structures are available for free TFIIH complex, TFIIH incorporated into the PIC, and TFIIH as part of the NER machinery (reviewed in (Yu et al, 2023)). The Core Module has a roughly horseshoe shape, with the TFIIH ATPases, Ssl2/XPB and Rad3/XPD, at the two ends.…”

“…In the PIC, the Tfb3/MAT1 alpha-helix is disconnected from Ssl2/XPB, producing an “open” conformation with the Ssl2/XPB rotated outward, its active site bound to the double-stranded DNA downstream of RNApII. TFIIH within the NER complex lacks the Kinase Module, and in this case the NER factors bring Rad3/XPD and Ssl2/XPB even closer together to facilitate the interaction of the damaged DNA with both ATPase active sites (Yu et al, 2023).…”

“…The apparent explanation for the existence of the “extra” subunits and functions in the TFIIH Core is that it also functions as a DNA nucleotide excision repair (NER) enzyme. In that process, both ATPases unwind DNA to expose a short stretch of the damaged strand for removal (Kim et al, 2023; van Eeuwen et al, 2021b; Yu et al, 2023). It therefore seems most likely that the TFIIH Core module originally evolved for DNA repair, and only later was co-opted for RNApII transcription.…”

Differing structures and dynamics of two photolesions portray verification differences by the human XPD helicase

“…The DNA entry pore, formed by the Arch domain together with the FeS and the ATPase lobe 1 domains, plays an important role in helicase activity, damage verification and NER efficiency ( 64 , 73–79 ). Several studies have yielded a number of significant insights on the role of the Arch dynamics in XPD’s helicase activity ( 67 , 74 , 80 , 81 ). Ghoneim and Spies showed that, when DNA bound, the Arch domain undergoes thermally driven open-close transitions that slightly favor the closed state with CPD present ( 74 ).…”