Function of Conserved Topological Regions within the Saccharomyces cerevisiae Basal Transcription Factor TFIIH

Abstract

bTFIIH is a 10-subunit RNA polymerase II basal transcription factor with a dual role in DNA repair. TFIIH contains three enzymatic functions and over 30 conserved subdomains and topological regions. We systematically tested the function of these regions in three TFIIH core module subunits, i.e., Ssl1, Tfb4, and Tfb2, in the DNA translocase subunit Ssl2, and in the kinase module subunit Tfb3. Our results are consistent with previously predicted roles for the Tfb2 Hub, Ssl2 Lock, and Tfb3 Latch regions, with mutations in these elements typically having severe defects in TFIIH subunit association. We also found unexpected roles for other domains whose function had not previously been defined. First, the Ssl1-Tfb4 Ring domains are important for TFIIH assembly. Second, the Tfb2 Hub and HEAT domains have an unexpected role in association with Tfb3. Third, the Tfb3 Ring domain is important for association with many other TFIIH subunits. Fourth, a partial deletion of the Ssl1 N-terminal extension (NTE) domain inhibits TFIIH function without affecting subunit association. Finally, we used site-specific cross-linking to localize the Tfb3-binding surface on the Rad3 Arch domain. Our cross-linking results suggest that Tfb3 and Rad3 have an unusual interface, with Tfb3 binding on two opposite faces of the Arch. TFIIH is a conserved 10-subunit complex that plays essential roles in both RNA polymerase II (Pol II) transcription and nucleotide excision repair (NER) (1-3). TFIIH and TFIIE are the last basal factors to join the transcription preinitiation complex (PIC) (4). In transcription, TFIIH functions in DNA unwinding, transcription start site (TSS) scanning, promoter escape, and Pol II C-terminal domain (CTD) phosphorylation. During NER, TFIIH promotes the DNA unwinding of an asymmetric region around the site of DNA lesions as an early step in excision and replacement of damaged DNA (5, 6). Mutations in three TFIIH subunits, XPD, XPB, and p8, cause human diseases associated with defects in DNA repair and transcription (1).TFIIH has three enzymatic activities that are essential for these functions. First, the Rad3/XPD (yeast/human) subunit is an ATPdependent DNA helicase that participates in DNA unwinding during NER. While this enzymatic activity is dispensable for transcription, Rad3/XPD is required for transcription as it plays an essential structural role in stabilizing the TFIIH complex (7-10). Second, Ssl2/XPB is an ATP-dependent double-stranded DNA translocase with roles in transcription and DNA repair. Ssl2 promotes formation of the Pol II open complex by reeling downstream promoter DNA into the Pol II active site, leading to DNA unwinding (11, 12). In budding yeast, Pol II transcription initiates downstream from the site of PIC formation (13), and Ssl2 function drives the scanning of downstream promoter DNA for an appropriate TSS (14). In the human transcription system, XPB activity has been implicated in promoter escape and the release of Pol II from contacts with promoter DNA and other PIC components (15, 16). Ssl2...

The cryo-electron microscopy structure of human transcription factor IIH

“…Combined with secondary structure prediction showing a very long α-helix in MAT1 (Extended Data Fig. 4) and CX-MS and site-specific crosslinking data showing proximity of MAT1 to XPB and XPD 7,29 (Fig. 3c, d), these observations strongly support our assignment of MAT1.…”

“…Mutations of C259 (partly occluded yellow sphere) impair the MAT1–XPD interaction. Site-specific crosslinks (SS) between RAD3 and TFB3 (yeast homologues of XPD and MAT1) 29 shown as purple spheres, CX-MS crosslinks (CX; same as in c ) light orange. Unassigned density that may correspond to the N-terminal RING domain of MAT1 (Extended Data Fig.…”

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

“…Consequently, the deletion of the C-terminal p34 domain affects both the stability of human TFIIH and its NER and transcription activities (Figure 4E and F ). Intriguingly, yeast genetics demonstrated that the deletion of the C4 domain was lethal ( 10 ). How the C-terminal domain of p34 exactly interacts with other TFIIH partners requires further investigation in future studies.…”

The complete structure of the human TFIIH core complex

“…15 residues of p44 span the 175 distance between the p44 vWFA domain and the XPB NTD, where a small helical 176 motif in p44 contacts XPB residues 72-75, 95-102, and 139-143, in agreement 177 with CX-MS data (Luo et al, 2015) (Figure 1 -figure supplement 6E). Partial 178 deletion of the p44 NTE in yeast causes a slow-growth phenotype, suggesting a 179 functional role for this p44-XPB interaction (Warfield et al, 2016). The XPB NTD is the site of the two human disease mutations F99S and T119P, 302 which cause XP and TTD, respectively (Cleaver et al, 1999).…”