Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kenji Murakami

Abstract

TFIIH is an essential, ten protein complex that necessary in both transcription initiation andNucleotide Excision Repair (NER). During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the C-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5 Å resolution cryo-EM structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologues of CDK7, Cyclin H, and MAT1, respectively. The C-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with previous cryo- EM structure of the pre-initiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK. The versatile NER pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo- EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3’ and 5’ side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification. These two structural studies have demonstrated some functional conservation in the mechanism of DNA unwinding between transcription and NER and provide the groundwork for further investigations into TFIIH function.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Additional Files

Movie 2.1.mp4 (66173 kB)
Movie 3.1.mp4 (24750 kB)
Movie 3.2.mp4 (68347 kB)
Movie 3.3.mp4 (46855 kB)
Movie 3.4.mp4 (29137 kB)

Share

COinS