Molecular Genetic Dissection of the RNA Polymerase II Transcription Factor D (TFIID) Subunit Taf2

Abstract

The evolutionarily conserved 14-subunit RNA Polymerase II Transcription Factor D (TFIID) complex is composed of TATA-box Binding Protein (TBP) and 13 TBP-associated factors (Tafs). In Saccharomyces cerevisiae, TFIID is essential for life as are each of its subunits; gene deletions and certain mutations result in cell inviability or temperature sensitive (Ts) growth phenotypes. Despite decades of study, the mechanisms by which many Taf subunits contribute to the essential function of TFIID are only poorly understood. In the accompanying dissertation, I address this gap in knowledge for two yeast TFIID subunits, Taf2 and Taf14. To understand the function of Taf2 in vivo, I performed a molecular genetic dissection of the TFIID subunit Taf2. Through systematic site-directed mutagenesis, I discovered a family of taf2ts alleles, two of which display growth defects that can be strongly suppressed by overexpression of the yeast-specific TFIID subunit TAF14 but not by overexpression of any other TFIID subunit. In S. cerevisiae, Taf14 is also a constituent of six other transcription related complexes making interpretation of its role in any of these complexes difficult. While Taf14 is not conserved as a TFIID subunit in metazoans, it is conserved through its chromatin binding YEATS domain. Based on my identification of the Taf2-Taf14 genetic interaction, I demonstrate that Taf2 and Taf14 directly interact and mapped the Taf2-Taf14 interaction domains. I used this information to identify a Taf2 separation-of-function variant (Taf2-ΔC). While Taf2-ΔC no longer interacts with Taf14 in vivo or in vitro, it stably incorporates into the TFIID complex. In addition, purified Taf2-ΔC mutant TFIID is devoid of Taf14 making this variant a powerful reagent for determining the role of Taf14 in TFIID function. Additionally, I developed methodologies for reconstitution of the TFIID complex using inducible overexpression in S. cerevisiae. This methodology could lead to important insights into the molecular architecture and assembly pathway of the TFIID complex. Successful reconstitution of TFIID using recombinant expression technologies could pave the way for in-depth genetic and biochemical dissection of the essential functions TFIID plays in transcriptional regulation.