Assembly of partial TFIID complexes in mammalian cells reveals distinct activities associated with individual TATA box-binding protein-associated factors

The TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s) co mpose the general transcription factor TFIID. The TAF(II) subunits mediate activated transcription by RNA polymerase II by interacting directly with s ite-specific transcriptional regulators. TAF(II)s also participate in promo ter recognition by contacting core promoter elements in the context of TFII D. To further dissect the contribution of individual TAF(II) subunits to ma mmalian TFIID function, we employed a vaccinia virus-based protein expressi on system to study protein-protein interactions and complex assembly. We id entified the domains of human (h) TAF(II)130 required for TAF(II)-TAF(II) i nteractions and formation of a complex with hTBP, hTAF(II)100, and hTAF(II) 250. Functional analysis of partial TFIID complexes formed in vivo indicate d that hTAF(II)130 was required for transcriptional activation by Sp1 in vi tro. DNase I footprinting experiments demonstrated that purified hTBP/hTAF( II)250 complex reconstituted with or without additional TAF(II)s was signif icantly reduced for TATA box binding las much as 9-fold) compared with free hTBP. By contrast, hTAF(II)130 stabilized binding of hTBP to the TATA box, whereas hTAF(II)100 had little effect. Thus, our biochemical analysis supp orts the notion that TAF(II)s possess distinct functions to regulate the ac tivity of TFIID.