RNA polymerase (RNAP) II catalyzes the transcription of protein-coding genes in eukaryotic cells. The largest subunit of RNAP II contains at its cis C-terminus an unusual domain consisting of multiple repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain (CTD) is extensively phosphorylated in RNAP IIO and unphosphorylated in RNAP IIA. Each round of transcription appears to be associated with reversible phosphorylation of the CTD. The studies proposed here will provide a critical test of the hypothesis that the CTD functions at multiple steps in the transcription cycle and that its activity is modulated by phosphorylation. This proposal is specifically concerned with an analysis of the CTD of mammalian RNAP II in the hope of better understanding its function and the consequences of the modifications that occur within it. The two primary objectives of this proposal are a) to define the interactions in which the CTD participates as RNAP II progress through the transcription cycle and b) to characterize the protein kinases and protein phosphatases that modulate the state of phosphorylation of the CTD. An understanding of the involvement of the CTD at specific steps in the transcription process is dependent on defining the proteins with which it interacts and how these interactions are influenced by phosphorylation. A novel approach to determining CTD interactions within the context of the transcription apparatus is to modify the CTD with a photoreactive probe and define these contacts by protein-protein crosslinking. CTD interactions with the transcription apparatus will also be defined by identifying transcription factors that mediate the differential interactions of RNAPs IIA and IIO with the preinitiation complex. An understanding of the regulation of CTD kinases and CTD phosphatase(s) that together control the extent of CTD phosphorylation is also fundamental to the understanding of how the activity of RNAP II is regulated. This proposal emphasizes the characterization and molecular cloning of the genes encoding two distinct CTD kinases, designed CTDK1 and CTDK2, that appear to recognize different target sequences within the consensus repeat. An analysis of CTDK1 and CTDK2 in conjunction with the tyrosine CTD kinase c-Abl will provide a test of the idea that phosphorylation of the CTD with different protein kinases can generate distinct conformations that differentially influence RNAP II activity. This proposal is also concerned with the characterization and molecular cloning of the gene(s) encoding a recently purified CTD phosphatase and an examination of how CTD phosphatase activity is regulated.
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