HUMAN T CELLS

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DESCRIPTION (adapted from applicant's abstract): Human Immunodeficiency virus type 1 (HIV-1) regulatory protein Tat increases the synthesis of full length RNA transcripts from the viral LTR. Tat interacts with transcripion factors and RNA polymerase II (RNAP II) enzyme complex to increase the efficiency of the transcript elongation process. One possible mechanism for Tat function proposes the association of Tat with specific kineses which phosphorylate the c-terminal domain (CTD) of the large subunit of RNAPII. The hypophosphorylated RNAPII is associated with the transcription initiation complex while the hyperphosphorylated RNAPII is associated with the elongation complex, and the regeneration of the hypophosphorylated form will be required for the next round of transcription. We reasoned that a Tat associated phosphatase may interact with Tat and RNAPII in the transcription complex to regenerate the hypophosphorylated form of RNAPII. We have partially characterized a Tat associated phosphatase which is sensitive to okadaic acid and decreases the CTD phosphorylation by a Tat associated CTD kinase (TTK). The long term goal of this project is to isolate the cDNA encoding the Tat associated phosphatase from human T cells and characterize the mechanism of its function in Tat trans-activation. The substrate specificity of the Tat associated phosphatase will be determined by using various phosphorylated proteins such as the large subunit of RNA polymerase II, recombinant c-terminal domain of RNAP II, dk7, and phosphorylase a. The domain requirement of Tat and the specificity of the phosphatase and Tat interaction will be studied by using mutants of Tat and an unrelated viral transactivator VP16. In viva association of the phosphatase with Tat will be studied by immunoprecipitating the Tat associated proteins from the cell Iysate of a HeLa cell line expressing HA-tagged Tat protein. Afterwards, the Tat associated phosphatase will be purified by chromatography. Antibodies will be generated using oligopeptides sepecific to the purified phosphatase. The cDNA encoding the phosphatase will be isolated by screening a cDNA expression library from T cells and its properties and functions characterized. These studies will not only help in gaining a better understanding of the mechanism of Tat response, they target issues relevant to basic mechanisms of RNA transcription.