Figure 1.

Repression of transcription by PC4 in the absence of TAFs and TFIIH. (A) Minimal transcription reactions contained 2 ng TBP (6), 10 ng TFIIB, 25 ng TFIIF, 50 ng pol II, and 50 ng of pMLΔ53 template DNA (containing the Ad ML core promoter). Either 20 ng (lanes 2 and 5) or 100 ng (lanes 3 and 6) of recombinant PC4 and recombinant TFIIE (lanes 4–6), composed of 5 ng TFIIEα and 2.5 ng TFIIEβ subunits, were added as indicated. (B) Transcription reactions were assembled as in A and contained TFIIE. Reactions in lanes 3 and 4 were supplemented with a fraction containing TFIIA. A total of 75 ng PC4 was added as indicated. (C) Transcription reactions with the pG5ML template, containing five GAL4 sites upstream of the Ad ML core promoter were carried out as in A with either TBP (4 ng, lanes 1–4 and 9–12) or an equivalent (normalized to its TBP content by Western blot analysis) amount of f:TFIID (lanes 5–8 and 13–16). PC4 (75 ng) was added to reactions in each even-numbered lane and activator (GAL4-AH, 40 ng) to reactions in lanes 3, 4, 7, 8, 11, 12, 15, and 16. Reactions 9–16 contained purified (Ni-NTA-agarose fraction) TFIIH. All reactions contained TFIIA and TFIIE. (D) Abortive initiation reactions with the dinucleotide CpA and [α-³²P]CTP employed pG5ML as template. Reactions in lanes 1–5 were reconstituted with TBP, pol II, TFIIB, TFIIE, and TFIIF. Reactions in lanes 6–9 were reconstituted with f:TFIID, TFIIA, pol II, TFIIB, TFIIE, TFIIF, and TFIIH. PC4 (lanes 4, 5, 7, and 9) and GAL4-AH (lanes 8 and 9) were added as indicated.