title: Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant.
abstract: Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeastSaccharomyces cerevisiaeThese include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1ASto map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We 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 abona fideAD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1ASvariant will likely also be useful for studies of the functions of Rap1 in other biological pathways.
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: Amanda N
authors.initials: AN
authors.lastname: Johnson
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 tony.weil@vanderbilt.edu.
authors.forename: P Anthony
authors.initials: PA
authors.lastname: Weil
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D000089804:Shelterin Complex; D035381:TATA-Binding Protein Associated Factors / Q000235:genetics / Q000378:metabolism; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; C440829:RAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; C438897:TAF5 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D034501:Telomere-Binding Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors
keywords: Saccharomyces cerevisiae; altered DNA binding specificity; gene regulation; protein engineering; transcription activation; transcription activator; transcription regulation; yeast