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-ÎC). While Taf2-ÎC no longer interacts with Taf14 in vivo or in vitro, it stably incorporates into the TFIID complex. In addition, purified Taf2-ÎC 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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-12132016-094213 |
| Date | 13 December 2016 |
| Creators | Feigerle, Jordan Taylor |
| Contributors | Roland Stein, William P. Tansey, Bryan Venters, Roger Colbran |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-12132016-094213/ |
| Rights | restrictone, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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