FIG. 2.

TAF40 interacts directly with TFIIA or TBP. (A) To test whether TFIIA interacts directly with TAF40 in vitro, a GST pull-down assay was used. Recombinant GST-TFIIA, GST-TBP, GST-TFIIB, or GST was incubated with recombinant histidine-tagged TAF40 (TAF40). After SDS-PAGE, TAF40 was detected using antibodies specific to the histidine tag of TAF40. Antibodies specific to the GST tag were used to confirm that similar amounts of GST and the GST fusion proteins were utilized (data not shown). (B) TAF40 increases TBP-TFIIA-DNA complex formation on a TATA box. For all reactions, 9 nM radiolabeled adenovirus early 1B TATA box probe was used. Concentrations of TBP and TFIIA were held constant at 5 and 1.5 nM, respectively. Lane 1 contains TBP, lane 2 contains TBP and TAF40 (142 nM), lane 3 contains TFIIA, lane 4 contains TFIIA and TAF40 (142 nM). Lane 5 contains TAF40 (142 nM) alone. Lane 6 contains TBP and TFIIA, and lanes 7 through 11 contain TBP and TFIIA with increasing amounts of TAF40 (19 to 142 nM). (C) TAF40 does not increase the TBP-TFIIB complex formation on a TATA box. All reactions are as in panel B, except that 3 nM TFIIB is used in place of TFIIA.