The CTD of RNA polymerase II is an unusual repeat domain that extends, tentacle-like, out from the globular body of the polymerase and acts as a scaffold to which nuclear factors bind; these associations physically link the factors to the transcription machinery. The CTD of elongating RNAP II is highly phosphorylated, as a result of the action of several CTD kinases, and it appears that different patterns of CTD phosphorylation effect recruitment of distinct factors to the transcribing polymerase. However, current understanding of the process of CTD phosphorylation is quite fragmentary. Moreover, while the functions of dozens of nuclear proteins are undoubtedly influenced by phosphoCTD binding, only a few phosphoCTD-associating proteins (PCAPs) have been characterized. Our overall goal is to generate extensive new information on CTD kinases and PCAPs that will bring about enhanced understanding of CTD phosphorylation and phosphoCTD function; we believe that this in turn will engender novel insights into a large number of fundamental nuclear functions and events. To achieve this goal we plan the following Specific Aims: ? ? 1. Determine the modes and consequences of CTD phosphorylation by CTD Kinase I. We will describe with biochemical rigor the disposition of phosphates on the CTD in vitro and in vivo. ? ? 2. Characterize binding domains and properties of novel PCAPs. We will identify phosphoCTD-interacting domains (PCIDs), then determine their phospho-epitope binding specifity and 3D structure. ? ? 3. Investigate and describe PCAP binding and functions in vivo. We will characterize association of PCAPs with target genes in vivo and assess how PCTD binding contributes to PCAP functions. Since the CTD and its transactions are highly conserved from yeast to man, this research is directly relevant to human biology and disease; it will contribute ultimately to betterment of human health. ? ?
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