The HIV-1 regulatory protein Tat plays a key role in the viral life cycle. Tat greatly increase the production of viral RNA by alleviating a defect in transcriptional elongation so that full-length viral transcripts can be generated. The mechanism by which this is accomplished is not well understood. Our recent studies have linked this increased efficiency of RNA chain elongation to a factor known as P-TEFb (positive transcription elongation factor b), newly-identified in human cells. P-TEFb is able to phosphorylate the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II in a reaction that is critical for transcriptional elongation. P-TEFb is related to, if not identical with, the CTD kinase TAK which is characterized by its ability to bind Tat. TAK and P-TEFb share at least one subunit, the CDC2-related kinase PITALRE. The major objectives of this grant are to evaluate the hypothesis that Tat acts via TAK/P-TEFb, and to extend it by examining the structure, function and regulation of these entities and characterizing their interactions with Tat. In addition, we propose to identify and study factor(s) responsible for potentiating Tat's ability to stimulate HIV-1 directed transcription in a cell-free system.
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