A large combinatorial assortment of DNA sequence-specific transcriptional activators control the expression of the vast repertoire of eukaryotic cellular and viral genes, which in turn dictate the biochemistry of the cell. How the multitude of activators funnel their signal into a common target, RNA polymerase II which transcribes these genes, is currently unknown. Solving this unknown would provide a major step toward understanding the biochemistry of cellular development differentiation, oncogenesis, and viral infectivity. This project proposes to chip away at the unknown biochemistry of transcriptional regulation by investigating the molecular mechanism through which one transcriptional activator, human Sp1, communicates with the human transcription machinery. In vitro assays which reconstitute Sp1-regulated transcription with purified components are already in place. The factors which Sp1 targets in the initiation complex have been localized to a purified multi-subunit TFIID complex. TFIID is a key component of a larger RNA polymerase II transcription initiation machine assembled at the promoter. The next step is to biochemically characterize purified intact TFIID complexes and their individual subunits (TAFs). Differences in subunit composition are proposed to generate multiple TFIID complexes, each with different gene regulatory functions. TFIID complexes have been and will be purified by immuno-affinity chromatography using antibodies directe against its TATA box binding subunit (TBP). First, the number of chromatographically distinct TFIID complexes and their subunit composition will be assessed by silver stained SDS polyacrylamide gels. Second, denaturants such as urea will be used to fractionate purified TAF subunits from each other, which will then be biochemically characterized. One or more TAFs comprise the SP1 coactivator which is necessary for Sp1-activated but not basal transcription. The purified coactivator will be reconstitute and its role in assembling an Sp1-regulated initiation complex addressed by DNase I protection, electrophoretic mobility shift, and kinetic assays. These assays will allow the following questions to be addressed: What rate limiting step in initiation does Sp1 target? Are on/off rates of limiting initiation factor affected? Does Sp1 target and mollify an inhibitory activity in the TFIID complex? An unusual feature of Sp1 is its ability to activate transcription at promoters which lack a TATA box. At such promoters an additional component of the TFIID complex, termed a tethering factor, is essential for Sp1- activated transcription; at TATA-containing promoters, Sp1-activated transcription does not require the tethering factor. The tethering factor does not appear to recognize the promoter directly through protein-DNA interactions, but is proposed to achieve promoter specificity through protein-protein interactions with Spl. AS with the coactivator the tethering factor will be isolated, cloned, and its role in assembling an initiation complex at a TATA-less promoter investigated. It is anticipated that information gained from this study will provide an essential missing piece to the gene regulation puzzle.
