The transcription initiation apparatus includes a set of general factors that associate with promoter DNA and with RNA polymerase II during initiation. The general transcription factor TFIID is the first to associate with promoter DNA during assembly of the initiation complex. The native form of the general transcription factor TFIID consists of the TATA binding protein (TBP) and multiple TBP-associated factors (TAFs) in higher eukaryotes, and we have recently found that this is also the case for yeast. This proposal is designed to identify and clone the genes for the complete set of Saccharomyces cerevisiae TBP-associated factors, and to use these genes to pursue molecular genetic and biochemical experiments designed to elucidate the structure of yeast TFIID and the roles of TAFs in transcription initiation. To accomplish these goals, the three specific aims of this proposal are: i) to isolate genes encoding TBP-associated factors in S. cerevisiae using genetic and biochemical approaches; 2) to investigate the effects of mutations on individual TAF functions in vivo; and 3) to study the function of TAFs and the TBP/TAF complex in vitro. To isolate genes encoding TAFs, we will continue to exploit a genetic selection that has already led to the isolation of four TAF genes; the four extragenic suppressors of RNA polymerase II CTD truncation mutants studied to date encode TAFs. As a biochemical approach to isolating TAF genes, we will identify the protein components of a high molecular weight complex containing TBP, subject these proteins to microsequence analysis, and use this information to design oligonucleotides for gene isolation. To investigate the effects of mutations on individual TAF functions in vivo, we will construct null and conditional mutations in the TAF genes and analyze their effects on transcription in vivo. To investigate the functions of the TAFs in the TFIID complex, wild-type and mutant TFIID preparations will be examined in a variety of transcription assays designed to assess the role of TAFs in template assembly and commitment and in basal and activated transcription. The health relatedness of this research derives from its contribution to the understanding of the basic molecular mechanisms that control gene expression.
