Biochemical and genetic analyses of interactions between transactivators and TBP associated factors in Saccharomyces cerevisiae

Abstract

The goal of my dissertation project was to characterize interactions between the transactivator Rap1 and Taf subunits of the TFIID complex. This experimental problem falls under a larger umbrella of gene regulation, since TFIID makes widespread contributions to Pol II transcription. The precise mechanisms underlying TFIID function remain obscure. A major clue to orient dissection of TFIID function is that transactivator proteins are known to directly interact with Tafs. However, there is little information on the physiological relevance of these interactions much less about precise effects on TFIID activity; this relates to difficulties in undertaking a multi-faceted experimental investigation in the metazoan organisms where transactivator-Taf interactions were first discovered. No such limitations exist in the baker’s yeast Saccharomyces cerevisiae, although at the time our studies were initiated the best characterized yeast transactivators were known to function by TFIID-independent mechanisms. A breakthrough occurred when it was shown that the yeast Rap1 activator and TFIID physically occupy and regulate common target genes, and that there exist regulatory interactions between these factors. Subsequently our lab identified and characterized physical interactions between Rap1 and TFIID, studies that I participated in. My initial involvement grew into this dissertation. To narrow my focus, I identified three Taf proteins that Rap1 interacts with. These three Tafs co-localize in the three-dimensional TFIID structure, an observation that persuaded me to consider that each Rap1-Taf interaction is physiologically relevant. To narrow my focus further, I defined the Rap1-binding domains within each Taf. I found that two of the three Taf domains are required for cellular viability, and that amino acid substitutions within either domain confer reduced cellular growth. Such cells exhibit deficiencies in transcription of most genes co-regulated by Rap1 and TFIID, but not at the expense of Taf or TFIID stability. Consistent with compromised Rap1-TFIID interaction, we were able to show that Tafs containing amino acid substitutions bind Rap1 with reduced affinity. I obtained evidence regarding specific Rap1 domains involved in Taf interaction. Finally, I conducted a molecular dissection of the Taf4 Rap1 binding domain, and identified two essential regions at amino acid resolution.