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Browsing Theses and Dissertations by Author "Almeida, Adam Raymond, author"

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    Spn1, a highly conserved and essential node of RNA polymerase II dependent functions
    (Colorado State University. Libraries, 2011) Almeida, Adam Raymond, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Woody, Robert, committee member; Suchman, Erica, committee member
    A multitude of proteins are responsible for regulating the activity of RNA Polymerase II (Pol II) in the nucleus of a eukaryotic cell. Two types of themes are used by these proteins to control transcription: recruitment-regulation and postrecruitment-regulation. The main difference between the two is the rate-limiting step for producing transcript. This rate-limiting step for the first mechanism is the recruitment of Pol II to the promoter. For the second mechanism, Pol II constitutively occupies the promoter, is "poised", and an unknown rate-limiting postrecruitment step prevents transcription from commencing. The highly conserved and essential transcription factor Spn1 was identified as a protein that functions postrecruitment of Pol II and has been characterized for having a direct role at regulating the poised CYC1 gene in Saccharyomyces cerevisiae. This activity has been determined from mutations made within the most conserved portion of Spn1 made up of a highly folded central domain. Little is known about the functions of the N-and C-terminal regions flanking this central domain, which is the focus of the work done here. Genetic characterization indicates that these regions have physiologically relevant and important functions within the cell outside of optimum growth conditions, but do not involve significant regulation of the CYC1 gene. A broader approach of experimentation is likely required to understand all of the Spn1 protein's functions regarding transcription. This led to the observation that Spn1 is able to bind to nucleosomes in vitro and that this interaction is dependent on the N-and C-terminal regions of the protein. The possibility that Spn1 could affect nucleosome dynamics in the cell is consistent with the physical and genetic interactions observed between Spn1 and the Spt6 and Swi/Snf histone chaperone and chromatin remodeling complexes. This result will provide several new avenues for future Spn1 research. A genomic ChIP-chip experiment performed by two independent groups revealed that Spn1 is recruited to a majority of the genes in the yeast genome. Evidence indicates that there are multiple, evolutionarily conserved pathways within the cell that are responsible for determining the rate at which an organism will age that include: ribosome biogenesis, protein translation, mitochondrial activity and function, heterochromatic stability, maintenance of the genome, and apoptosis. The possibility that Spn1 regulates the genes involved in these pathways is highly suggestive that this protein could be an aging factor within the cell. Chronological aging assays revealed that the removal of the N-and C-terminal regions of the Spn1 protein dramatically increase the lifespan of the BY4741 strain of yeast. These results further verify the physiological importance of this protein and the need for further Spn1 research.