Figure 2.

Effect of TAF_IIs on transcriptional activation by Dorsal in vitro. (A) Ability of TBP versus TFIID to mediate activation by Dorsal in a purified in vitro transcription system. (Left) In vitro transcription reactions containing the basal factors and RNA polymerase II but lacking TBP or IID (lanes 1–3) were programmed with 3 × DlTwiE1BCAT as template in the absence (lanes 1, 4, and 7) or presence of Dorsal (10 ng, lanes 2, 5, and 8 or 40 ng, lanes 3, 6, and 9) supplemented with TBP (lanes 4–6) or TFIID (lanes 7–9) and were assayed by primer extension. (Center) Coomassie blue-stained SDS polyacrylamide gel of purified Dorsal (lane 11). (Right) Silver-stained SDS polyacrylamide gel of purified TFIID (lane 12). The molecular sizes of protein standards (in kilodaltons) are indicated on the left. The identity of the TAF subunits is indicated on the right of lane 12. (B) Dorsal interacts with dTAF_II110 and dTAF_II60. Anti-FLAG antibody resin (lanes 2, 5, 8, 11, 14, and 17) or beads loaded with Dorsal (lanes 3, 6, 9, 12, 15, and 18) were incubated with the [³⁵S]methionine-labeled, in vitro-translated TAFs indicated at the bottom of each panel. Protein complexes were analyzed by SDS/PAGE. Bound TAFs were identified by autoradiography. Lanes 1, 4, 7, 10, 13, and 16 represent 10% of the input material for each binding reaction.