Fig. 3.

Dissecting higher order complexes with aptamers. (A) Different sensitivity of three- and four-component complexes to different TBP-aptamers. All reactions contained 0.5 nM radioactive TATA-DNA (indicated by an asterisk) and 2.5 nM His-yTBP. In addition, 2.5 nM TFIIA (lanes 1–6), 100 nM TFIIB (lanes 7–12), or both (lanes 13–18) were included to form higher order complexes. Two different concentrations of unlabeled RNA or TATA-DNA, as indicated to the left of the gels, were added after a 30-min incubation to disrupt the preformed complexes. The mixtures were incubated for another 30 min before loading onto the gel. (B) Prevention of TFIIA·TFIIB·TBP·TATA complex formation by aptamers. Reactions with same conditions in A except that 40 nM unlabeled RNA or TATA-DNA was added together with the radioactive probe (lanes 9–14). Lanes 1–8 are controls showing the process of TATA-dependent complex formation in the absence of RNA aptamers.