FIG. 7.

Requirement of GTFs for HPV-11 E2-mediated repression of the homologous E6 promoter. (A) E2-mediated repression in a highly purified in vitro transcription system. In vitro transcription was conducted with recombinant TFIIB, TBP, TFIIE, TFIIF, and FLAG-tagged TFIIH and FLAG-tagged pol II as described in Materials and Methods, in the absence (−) or presence of increasing amounts of E2. Relative intensity in each set of reactions is defined as the ratio of the HPV signal, which is quantitated first by PhosphorImager and then normalized with the internal control, obtained in each reaction to that performed in the absence of E2 (i.e., the first lane of each reaction set). (B) TFIIE and TFIIH are not required for transcription from supercoiled HPV DNA templates. Transcription reactions were performed as described for panel A. The transcription components indicated above the lanes were then left out from the complete reaction (All). The subunits of TFIIF, RAP30 (F₃₀) and RAP74 (F₇₄), were also selectively left out from the complete reaction. (C) E2-mediated repression of the E6 promoter can be observed in a minimal transcription system containing only TBP, TFIIB, TFIIF, and pol II. In vitro transcription reactions were performed with TBP, TFIIB, TFIIF, and pol II, with or without (−) 50 ng of E2 and in the absence (M) or presence (C) of both TFIIE and TFIIH.