The mechanisms by which eukaryotes regulate gene expression are important to understand many complex biological phenomena including human diseases. The molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate several basic issues concerning molecular mechanisms of transcriptional regulation in yeast cells by combining chromatin immunoprecipitation (ChlP), molecular genetics, and biochemistry. First, mechanisms of activator-specific recruitment of TFIID and growth-regulation of ribosomal protein (RP) genes by Rap1 will be studied. We will define the Rap1-containing activator in molecular terms, address whether and how this activator directly interacts with TFIID, and define the factor(s) responsible for RP regulation by growth signals and protein kinase A. Second, to elucidate how TBP function at promoters is regulated, we will analyze NC2 and Mot1, which interact with TBP and are recruited to active promoters. We will identify the components of the Pol II machinery required for NC2 and Mot1 recruitment, determine whether and how the 3 TBP complexes compete for TATA occupancy in vivo, and use sequential ChlP to determine cooccupancy of Mot1, NC2, TFIID with each other and with components of the Pol II machinery. Third, we will use ChlP to address activator-specificity for recruiting different chromatin-modifying activities and targets in the Pol II machinery, ask whether DNA looping occurs in vivo via interactions between activators and the Pol II machinery, and use a novel strategy to assess basic parameters of DNA looping in vivo. We will also address the atypical activation mechanism used at the cyc1 promoter. Fourth, using mutants and chromatin structure assays, we will determine the relationship between nucleosome positioning and preferential accessibility of promoter regions in vivo, and ask whether promoter regions are really free of nucleosomes. Using in vitro chromatin assembly, we will ask whether intrinsic nucleosome positioning and/or nucleosome remodeling accounts for preferentiai accessibility. Fifth, we will identify the factors necessary to recruit the RSC nucleosome-remodeling complex to essentially all Pol lII promoters and to specific Pol II promoters in a manner that correlates with activation or repression. In addition, we will determine the kinetic stabilily of the nucleosome-remodeled state in vivo. Sixth, As DNA-binding specificity per se does not account for how proteins associate with genomic sequences in vivo, we will use ChlP and microarray technology to examine DNA-binding specificity of Gcn4 in vitro and in vivo on a genome-wide level, and determine how bZIP proteins with indistinguishable DNA-binding specificities in vitro have different target genes in vivo. We will also examine association of Hog1 MAP kinase to intergenic regions on a genome-wide level as a means of identifying direct targets of protein kinases involved in transcriptional regulation. Seventh, having developed two in vivo elongation assays, we will address whether potential elongation factors actually travel with elongating Pol II in vivo, and whether such putative elongation factors and putative elonrgation factors and inhibitors actually affect elongation or transcriptional arrest in vivo.
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