Many important biological processes such as cell growth, differentiation, and transformation are controlled by differential gene expression, which is regulated mainly at the level of transcription. The longer range objective of this proposal is to understand the molecular mechanisms by which genes important for such processes are activated and regulated. Related to this, the major goal is to understand the structure-function relationships of the yeast TATA box-binding protein (TFIID). This is a key transcription initiation factor which both binds to the TATA box to promote functional interactions of RNA polymerase II (and other general initiation factors) and serves as a target for the stimulatory (and possibly inhibitory) action of gene specific regulatory proteins. Toward this goal the specific aims are: (i) to map the domains in the protein responsible for specific DNA binding, and to elucidate specific base and amino acid contacts, (ii) to investigate the possible interactions of TFIID with each of the other general initiation factors involved in the assembly of preinitiation complexes, including analysis of the domains involved, (iii) to map the domains of TFIID involved in mediating the action of specific activators and repressors, including identification of any intermediary proteins that may be involved, and (iv) to determine possible modifications in TFIID structure and function, or the induction of distinct species of TFIID, in responses to changes in cell growth and differentiation. The techniques to be employed include site-directed mutagenesis and expression of cloned TFIID, functional analysis in cell free systems reconstituted with purified general and regulatory factors, and a variety of biochemical (high resolution footprinting, photo-affinity crosslinking, etc) and immunological techniques for analyses of DNA-protein and protein-protein interactions. Thus these analyses will provide novel information regarding the transfer of information from regulatory factors to the complex machinery which actually effects transcription of target genes. As there is virtually no information on this final critical step in any signal transduction pathway, this work should be generally relevant to the action of a broad variety of stimuli which ultimately affect gene activity. Additionally, a better understanding of the complex array of interactions, on the DNA, between regulatory factors and general factors should facilitate the discovery of ways to selectively control genes in both normal and abnormal (diseased) situations.
