The proposed research will be a systematic biochemical investigation of the proteins and DNA sequences required for transcription initiation in vitro by mammalian RNA polymerase II. The strategy involves a physical and functional characterization of protein-DNA complexes that represent intermediates in the assembly of initiation complexes at the promoter. A major focus of the research will be an extensive analysis of the interaction of the yeast and human TFIID proteins with the TATA box sequence element, as this step has been proposed to be a major determinant of promoter strength both in vitro and in vivo. Using a combination of experimental approaches, including chemical and enzymatic interference methods, gel mobility shift assays, and transcription reactions, the kinetics and stability of TFIID binding to different TATA box sequences will be measured; the role of individual bases within and flanking the TATA box will be probed; and the possible importance of DNA structure to this interaction will be examined. Similar experimental strategies will be used to analyze the protein-protein and protein-DNA interactions that contribute to the binding and function of other general transcription factors in the assembling preinitiation complex. In the initial phases of the research, transcription factors IIA and IIB will be of particular interest, because each of these proteins is capable of forming a complex with TFIID at the promoter in the absence of other proteins. The results from the quantitative and qualitative analyses of protein-DNA complexes formed with different promoter sequences will be correlated with measurements of the ability of these same promoters to function in transcription initiation in the presence and absence of upstream activating proteins. These studies are expected to provide a detailed understanding of the structure and function of the minimal class II promoter, to suggest mechanistic roles for the various proteins required for transcription initiation, and to define the steps that are rate-limiting for transcription. The long-term objective of this research is to identify the mechanistic steps in the assembly and initiation of transcription complexes that are targets of regulation and to determine how this regulation is accomplished.
