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Title page for ETD etd-05302017-100556


Type of Document Dissertation
Author Meyer, Amanda N.
URN etd-05302017-100556
Title Mechanisms of Transcription Activation by the DNA-binding Transfactor Repressor Activator Protein 1 (Rap1) Uncovered Using an Altered DNA-binding Specificity Variant
Degree PhD
Department Molecular Physiology and Biophysics
Advisory Committee
Advisor Name Title
P. Anthony Weil Committee Chair
Bryan Venters Committee Member
Roger Colbran Committee Member
Roland Stein Committee Member
William P. Tansey Committee Member
Keywords
  • gene regulation
  • protein engineering
  • Saccharomyces cerevisiae
  • transcription regulation
  • yeast
  • transcription activator
Date of Defense 2017-05-01
Availability restricted
Abstract
The enhancer DNA-binding transfactor Repressor activator protein 1 (Rap1) performs several essential cellular functions in the budding yeast Saccharomyces cerevisiae. These functions include regulation of telomere length, transcription repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of protein-encoding genes, including the ribosomal protein- and glycolytic enzyme-encoding genes. Rap1 was discovered over 30 years ago, and studies of Rap1-dependent transcription repression and telomere length stabilization have produced significant mechanistic insights. By comparison, the mechanism of Rap1 transcription activation remains poorly understood for two reasons. First, Rap1 is encoded by a single copy essential gene and its involvement in many disparate cellular functions prevents easy interpretation of direct Rap1 dissection studies. Second, the existence of conflicting reports of the ability of Rap1-heterolgous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenges the use of this conventional approach to study Rap1. In the accompanying dissertation, I address these challenges and uncover Rap1 contributions to transcription activation through the generation of an altered DNA-binding specificity variant of Rap1 (Rap1AS). Rap1AS possesses true altered DNA-binding specificity in that the mutant enhancer recognition it has gained is accompanied by a loss in recognition of the wild-type enhancer. Rap1AS is thus an ideal tool for dissecting Rap1 structure-function relationships. Using Rap1AS, I mapped and characterized a 41-amino acid activation domain (AD) within The Rap1 C-terminus. I found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. Rap1 AD mapping and identification of Taf5 as a coactivator target represent important advances in our understanding of how Rap1 contributes to gene-specific activation and will enable future dissection of Rap1-dependent transcription activation mechanisms.

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