The Role of Phosphorylation in Modulating PC4 Function as a General Cofactor

Purification of multisubunit protein complexes such as human TFIIH and RNA polymerase II has been a tedious task by conventional chromatography. To overcome the difficulty in isolating human TFIIH and RNA polymerase II, which play an essential role in eukaryotic transcription, we have developed an effective purification scheme that allows TFIIH and RNA polymerase II to be purified from HeLa-derived clonal cell lines that conditionally express the FLAG-tagged p62 subunit of human TFIIH and the RPB9 subunit of human RNA polymerase II, respectively. An approximate 2000-fold enrichment of the FLAG-tagged TFIIH and a 1000-fold enhancement of total RNA polymerase II are achieved by a one-step immunoaffinity purification. The purified complexes, which are devoid of other general transcription factors (GTFs) commonly copurified with TFIIH and RNA polymerase II by traditional approaches, are fully functional in mediating both basal and activated transcription, regardless of whether TBP or TFIID is used as the TATA-binding factor. Interestingly, repression of basal transcription by the positive cofactor PC4 is alleviated by increasing amounts of TFIID, TFIIH and a preassembled RNA polymerase II holoenzyme complex but not by the other GTFs or RNA polymerase II. Our finding suggests that phosphorylation of PC4 by TFIID, TFIIH or RNA polymerase II holoenzyme may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermore, transcriptionally active RNA polymerase II complexes with different phosphorylation states on the carboxy-terminal domain (CTD) of the largest subunit can be selectively purified from the inducible RNA polymerase II cell line, making it possible to dissect the role of CTD.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Kershnar, Eric Roger

Contributors dc:contributor

• Cheng-Ming Chiang

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI9990038

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/84919