FIG. 7.
TFIIA assembly at the promoter is BRE dependent. (A) The wild-type AdML promoter and mutant derivative AdMLmBREud were radiolabeled, and assembly of TBP and TFIIA was monitored by band shift analysis. Where indicated, 50 ng of human TBP (hTBP) was included in the reaction mixture. Either 50 ng or 250 ng of TFIIA was added. Free probe and TBP-TFIIA-promoter complexes are indicated at the right. (B) Same as panel A except that 10 ng and 50 ng of purified recombinant NC2 were included instead of TFIIA. Also, where indicated 500 ng of anti-NC2α antibody was included, and the supershifts are indicated. (C) TFIIA (50 ng) or NC2 (50 ng) was incubated with the radiolabeled AdML promoter either alone or along with hTBP (50 ng). Complexes were resolved as for panel A. TFIIA-TBP-AdML and NC2-TBP-AdML complexes and free probe are indicated. (D) Same as panel A except that 50 ng or 250 ng of recombinant human TFIIB was used instead of TFIIA.