Figure 3.

(A) Schematic diagram of the domain structure of wild-type and mutant dTAF_II110 and dTAF_II60. The black boxed area represents the dTAF_II250 interaction domain of each TAF. The dark gray area (marked coactivator) delineates the domains that are thought to interact with activator proteins. (B) TAF_II110ΔC, TAF_II110ΔB, and TAF_II60YY retain the ability to bind selectively to Dorsal. Anti-FLAG antibody resin (lanes 2, 5, and 8) or beads loaded with Dorsal (lanes 3, 6, and 9) were incubated with [³⁵S]methionine-labeled, in vitro-translated mutant TAFs indicated at the bottom of each panel. Protein complexes were separated by SDS/PAGE. Bound mutant TAFs were identified by autoradiography. Lanes 1, 4, and 7 represent 10% of the input material for each binding reaction. (C) Mutant dTAF_II110 and dTAF_II60 squelch activation by Dorsal but not basal transcription. Reactions containing the basal factors, RNA polII, 3 × DlTwiE1BCAT DNA template, and 0 ng of Dorsal (lane 1 and lanes 7–11) or 40 ng of Dorsal (lanes 2–6) were assayed in the presence of increasing amounts (30 ng or 100 ng) of either TAF_II110ΔC (lanes 3 and 8 and lanes 4 and 9, respectively) or TAF_II60YY (lanes 5 and 10 and lanes 6 and 11, respectively). Coomassie blue-stained SDS/PAGE gels of mutant TAF_II110ΔC (lane 13) and TAF_II60YY (lane 15) as well as molecular weight markers (lanes 12 and 14) are shown.