The long term objectives of this project are to understand how the process of elongation of transcription is regulated in cells and on the Human Immunodeficiency Virus type 1 Long Terminal Repeat (HIVLTR). The general strategy is to identify roles played by different Positive Transcription Elongation Factor b (P- TEFb) complexes, HEXamethylene blsacetiMide-inducible proteins 1 and 2 (HEXIM1/2), 7SK small nuclear RNA (7SK RNA), nuclear factor kappa B (NF-KB), HIV transcriptional Transactivator (Tat) and TransActivation Response (TAR) RNA in this process. P-TEFb contains the Cyclin dependent kinase 9 (Cdk9) and a C-type cyclin, CycT1, CycT2 or CycK. NF-KB contains p65 (RelA) and p50. Of special importance is the balance between inactive, high molecular weight (HMW) P-TEFb complexes that contain additionally 7SK RNA and HEXIM1/2 and active, low molecular weight (LMW) P-TEFb complexes that contain only Cdk9 and a C-type cyclin.
The specific aims are to define as precisely as possible surfaces that form inactive, HMW P-TEFb and active, LMW P-TEFb complexes that bind NF-KB and Tat. Two conformational states of HEXIM1/2 exist in cells, both oligomeric, one complexed with 7SK RNA and P-TEFb, the other free in solution. In addition, we want to determine the precise three-dimensional structure of the complex between CycT1, Tat and TAR, with special emphasis on surfaces on these proteins that bind RNA and the now stabilized TAR. Next, we shall investigate the function of these different P-TEFb complexes and how they are regulated by posttranslational modifications. Of special interest is the phosphorylation and ubiquitylation of HEXIM1/2. Once activated, how is P-TEFb recruited by NF-KB to the HIVLTR? What role does the ubiquitylation of p65 play in this process? From other studies, we know that monoubiquitin binds the C-terminus of CycT1. These studies will lead to the creation of constitutively active HEXIM1/2 proteins, which will be used to determine their differential effects on cellular and viral transcription. Finally, we shall compare the recruitment of P-TEFb by NF-KB via DNA and Tat via RNA in vitro and in vivo, study the dynamic interplay between Negative Elongation Factor (N-TEF) and P-TEFb on the HIVLTR by chromatin immunoprecipitation in cells as well as inactivate genes encoding CycT1, CycT2 and CycK in the mouse. Unique and redundant roles of P-TEFb complexes containing these C-type cyclins will be revealed in the organism. These studies will reveal the complex RNA-protein world of P-TEFb and how HIV subverts these cellular complexes for its replication in cells.
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