Immunoaffinity purification and functional characterization of human transcription factor IIH and RNA polymerase II from clonal cell lines that conditionally express epitope-tagged subunits of the multiprotein complexes

Purification of multiprotein complexes such as transcription factor (TF) LI N and RNA polymerase II (pol II) has been a tedious task by conventional ch romatography. To facilitate the purification, we have developed an effectiv e scheme that allows human TFIIH and pol II to be isolated from HeLa-derive d cell lines that conditionally express the FLAG-tagged p62 subunit of huma n TFIIH and the RPB9 subunit of human pol II, respectively. An approximate 2000-fold enrichment of FLAG-tagged TFIIH and a 1000-fold enhancement of to tal pol II are achieved by a one-step immunoaffinity purification. The puri fied complexes are functional in mediating basal and activated transcriptio n, regardless of whether TATA-binding protein or TFIID is used as the TATA- binding factor. Interestingly, repression of basal transcription by the pos itive cofactor PC4 is alleviated by increasing amounts of TFIID, TFIIH, and pol II holoenzyme, suggesting that phosphorylation of PC4 by these protein s may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermo re, pol II complexes with different phosphorylation states on the carboxyl- terminal domain of the largest subunit are selectively purified from the in ducible pol II cell line, making it possible to dissect the role of carboxy l-terminal domain phosphorylation in the transcription process in a highly defined in vitro transcription system.