Skip to main content
. 1999 Mar 2;96(5):1959–1964. doi: 10.1073/pnas.96.5.1959

Figure 2.

Figure 2

TRAP-mediated TR activation does not require TAFIIs. (A) Activation by the f:TRα/TRAP complex in the absence of TAFIIs. Reactions contained RNA polymerase II and general initiation factors as in Fig. 1, except that f:TFIID (containing 4 ng TBP) was replaced with TBP (4 ng) in lanes 1–4. Other additions, as indicated, included f:RXRα (20 ng), f:TRα (12 ng) plus TRIAC (2 μM), f:TRα/TRAPs (12 ng f:TRα, 80 ng total protein including TRAPs), and USA (250 ng). Quantitative immunoblot analyses revealed the presence of low amounts of TAFIIs in the TFIIE/TFIIF/TFIIH preparation, but the amounts contributed to the reactions were only 5–10% of the amounts contributed by the f:TFIID preparation and were insufficient to mediate normal high-level activation by Gal4-p65 (C); furthermore, f:TRα/TRAP-mediated activation was also observed, albeit at a somewhat reduced level, with a transcription complementation system that is TFIID/TAFII-free by both functional and Western blot analyses (data not shown). (B) Quantitation of TR in f:TRα and f:TRα/TRAP preparations. Purified f:TRα (12 ng) and immunopurified f:TRα/TRAP complex (12 ng of f:TRα, 80 ng of total protein including TRAPs) were fractionated by SDS/PAGE and assayed by Western blot analysis using mouse monoclonal antisera against the FLAG epitope. (C) Activation by Gal4-p65 requires TAFIIs. Reactions contained the same amounts of RNA polymerase II and general transcription factors as in Fig. 1, except that f:TFIID (containing 4 ng of TBP) was replaced by TBP (4 ng) in lanes 1 and 2, in addition to USA (250 ng) and f:Gal4-p65 (25 ng), as indicated. A template containing Gal4 binding sites fused to the adenovirus ML core promoter replaced the TR-responsive promoter (18). (D) Dose response of f:TRα-mediated activation in the presence and absence of TRAPs. Variable amounts of f:TRα (6 ng, lanes 2 and 6; 12 ng, lanes 3 and 7; 24 ng, lanes 4 and 8) were added to transcription reactions containing either 4 ng TBP (lanes 1–4) or an amount of highly purified f:TFIID containing 4 ng TBP (lanes 5–8) and other components, as indicated, at the concentrations employed in Figs. 1 and 2A.