FIG. 3.
E2-mediated repression of the HPV-11 E6 promoter in a reconstituted two-component transcription system. (A) TAFIIs are not required for E2-mediated repression. In vitro transcription was performed with TFIID-deficient pol II holoenzyme (f:pol II [67]) and either FLAG-tagged TFIID (f:TFIID [9]) or FLAG-tagged TBP (f:TBP), in conjunction with general cofactor PC4 (25, 35), in the absence (−) or presence of different amounts of E2. Unless otherwise specified, baculovirus-expressed FLAG-tagged HPV-11 E2 was used in the assay. (B) PC4 and phosphorylation on E2 are not required for E2-mediated repression. In vitro transcription was performed as described for panel A, except that E2 purified from either insect cells (Sf9) or bacteria (E. coli) was used and no PC4 was included in the experiment. (C) The initiator element and CEI and CEII are not required for E2-mediated repression. In vitro transcription was performed as described for panel A, except that different HPV-11 DNA templates were used. (D) E2 mainly acts through the no. 4 E2-binding site to inhibit E6 promoter activity. DNA templates containing either wild-type (WT) or mutated E2-binding sites were constructed in the backbone of p7862-70GLess/I− as described in Materials and Methods. 4M, 3M, 2M, 23M, 24M, 34M, and 234M are DNA templates containing mutations on the no. 4, no. 3, no. 2, no. 2 and 3, no. 2 and 4, no. 3 and 4, and no. 2 and 3 and 4 E2-binding sites, respectively. In vitro transcription was performed as described for panel A, except that different HPV-11 DNA templates were used.