Abstract
The TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s) compose the general transcription factor TFIID. The TAF(II) subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAF(II)s also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAF(II) 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) TAF(II)130 required for TAF(II)-TAF(II) interactions and formation of a complex with hTBP, hTAF(II)100, and hTAF(II)250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAF(II)130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAF(II)250 complex reconstituted with or without additional TAF(II)s was significantly reduced for TATA box binding (as 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 supports the notion that TAF(II)s possess distinct functions to regulate the activity of TFIID.
Highlights
Regulation of transcription in eukaryotes requires the participation of a number of transcription factors, many of which exist as multiprotein complexes
The Conserved C-terminal Domain of hTAFII130 Is Required for Stable Association with the Endogenous TFIID Complex in Transfected 293T Cells—We previously demonstrated that recombinant hTAFII130 transiently expressed in 293 cells can stably associate with the endogenous TATA box-binding protein (TBP) and TAFIIs to form a TFIID complex [32]
The presence of endogenous TAFIIs in immunopurified TFIID was detected by probing the same immunoblots with ␣-hTAFII250 antibody and found hTAFII250 to be present in all lanes immunoprecipitated with ␣-hTBP antibody
Summary
TFIID, we have carried out biochemical analyses utilizing the vaccinia virus protein expression system [29]. The transient transfection protocol allowed us to test a series of mutant constructs of hTAFII130 for TAFII-TAFII interactions and their ability to assemble into a partial complex with other subunits without the need to make recombinant viruses for each mutant. The recombinant viruses generated were used for purification of dimeric, trimeric, and tetrameric complexes. Using these vaccinia virus-based assays, we examined the assembly among TBP and different TAFII subunits and mapped the “surfaces” required for these interactions. The functional analysis of individual TAFII subunits both separately and in the context of the partial TFIID complexes has permitted us to assess the role of each subunit in TFIID function
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