We have identified a protein kinase activity which adds multiple phosphate groups to the carboxyl-terminal repeat domain (CTD) of the largest subunit of eukaryotic RNA polymerase II. We plan to purify and characterize this kinase from both yeast and Drosophila and to clone and sequence the gene encoding it. We will then use this information and the genetic manipulations available to yeast and Drosophila to help elucidate the physiological roles of the kinase, focusing in particular on how CTD phosphorylation modulates the properties of RNA polymerase II. Knowledge gained from these investigations, by revealing mechanisms regulating functional properties of the transcription apparatus, will contribute to our understanding of gene expression in all eukaryotes.
SPECIFIC AIMS : 1. Complete the purification of the kinase. 2. Prepare antibodies against the purified enzyme. 3. Clone the kinase gene from yeast and Drosophila using the antibodies and expression vector Libraries. 4. A. Sequence the yeast gene; compare the deducted amino acid sequence to known kinases and oncogenes. B. Disrupt the yeast gene, reintroduce it into the genome, and investigate the null phenotype (anticipate null-lethal). C. Construct conditional mutants; use them to investigate consequences of inactivating the kinase (especially effects on RNA polymerase II). 5. Map the Drosophila gene by in situ hybridization; attempt to correlate it with a known genetic locus. 6. Generate mutants in the Drosophila locus (or use pre-existing ones); investigate their phenotypes and effects on RNA polymerase II.
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