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

Abstract

The TATA box-binding protein (TBP) and TBP-associated factors (TAFIIs) compose the general transcription factor TFIID. The TAFII subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAFIIs also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAFII subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAFII130 required for TAFII-TAFII interactions and formation of a complex with hTBP, hTAFII100, and hTAFII250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAFII130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAFII250 complex reconstituted with or without additional TAFIIs was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAFII130 stabilized binding of hTBP to the TATA box, whereas hTAFII100 had little effect. Thus, our biochemical analysis supports the notion that TAFIIs possess distinct functions to regulate the activity of TFIID.