We plan to continue our investigations of novel protein kinases that hyperphosphorylate the unique C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. The phosphorylation of this domain is a process that has been conserved from yeast to man, and our investigations are aimed at elucidating the physiological role played by CTD phosphorylation and at identifying and characterizing the enzymes involved in this unusual modification of the transcription apparatus. Knowledge gained from these investigations should uncover new enzymatic and regulatory activities and reveal mechanisms regulating functional properties of the transcription machinery. By contributing to our understanding of gene expression in all eukaryotes, these studies will provide an increased ability to understand basic cellular processes that may be disturbed in certain disease states.
Specific Aims : 1. Characterize more thoroughly, both enzymologically and genetically, the CTD-kinase we previously identified and purified from the yeast Saccharomyces cerevisiae. Manipulate the CTK1 gene for the alpha subunit of CTD-kinase to investigate possible roles of two unusual domains it possesses. 2. Clone, sequence and manipulate the genes encoding the beta and gamma subunits of yeast CTD-kinase. Use the information obtained to formulate ideas about the functions of these subunits. 3. Identify and analyze novel CTD kinase-related activities and their genes (""""""""CKR genes""""""""). Identify CKR genes by isolating synthetic lethals or extragenic suppressors of ctk1 mutations. 4. Use mutants in CTK and CKR genes to investigate patterns and consequences of CTD phosphorylation in vivo and in vitro. 5. Investigate CTD kinases and related activities in Drosophila. Using information and reagents (e.g. clones, antibodies, substrates) generated by the studies in yeast, extend the investigation of CTD phosphorylation to a metazoan with facile genetics, Drosophila. 6. Investigate the effects of phosphorylation on CTD structure.
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