Title page for ETD etd-07062012-081210

Type of Document Master's Thesis
Author Le Blanc, Kimberly
URN etd-07062012-081210
Degree Master of Science (M.S.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
David Donze Committee Chair
Anne Grove Committee Member
Patrick DiMario Committee Member
  • ETC sites
  • Insulator
  • Transcription
  • TFC6
  • Mis-expression
  • Auto-regulation
Date of Defense 2012-06-15
Availability unrestricted
RNA Polymerase III (Pol III) is best characterized for its transcription of tRNA molecules. Interestingly, Pol III and its associated complexes have been found to be involved in the regulation of transcription of genes transcribed by RNA Polymerase II (Pol II) (Kleinschmidt, LeBlanc et al. 2011). Extra TFIIIC sites (ETC sites) are chromosomally located sites discovered in S. cerevisiae that bind the Pol III transcription factor TFIIIC only through B-box interactions. These chromosomal locations are not normally transcribed (Dieci and Sentenac 1996). One of the subunits of the TFIIIC complex, TFC6 has an ETC site (ETC6) in its promoter region (Simms, Dugas et al. 2008; Kleinschmidt, LeBlanc et al. 2011). The TFIIIC complex has been shown to directly regulate transcription from the TFC6 promoter. This is a unique example of autoregulation of a Pol II transcribed gene by a Pol III transcription factor. Mutating ETC6 in the TFC6 promoter shows that both it and a mutation in TFC3, results in increased transcription of TFC6. Both mutations inhibit TFIIIC binding to ETC6. TFIIIC binding to ETC6 is inversely proportional to TFC6 transcription levels. Thus, when Tfc6p was overexpressed, promoter activity was inhibited. Via stringent control on Tfc6p levels, this autoregulation is hypothesized to be involved in global regulation of tRNA gene expression and on global regulation of translation. The previous results from Kleinschmidt et al. 2011 demonstrated that TFIIIC binding is altered by overexpressing TFC6. To examine whether the varied expression of Tfc6p has a global effect on TFIIIC binding, and possibly Pol III transcription, we created a set of yeast strains with significant variation in Tfc6p expression: wild type, under-expressed, and two levels of over-expression (two-fold and ~12-fold). By performing a genome wide chromatin immunoprecipitation analysis of TFIIIC binding when TFC6 expression is mis-regulated using high throughput sequencing (ChIP-Seq) technology, we expect that different TFIIIC bound loci will show variations in the ChIP-Seq signals that will differ in magnitude. This analysis will allow us to asses how TFC6 mis-regulation effects tDNA transcription at those loci most sensitive to TFC6 mis-regulation, it may reveal cryptic ETC sites and reveal any changes in the extra-transcriptional functions of Pol III.
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