The main goal of the proposed research is to understand the architecture, dynamics, and function of complex assemblies involved in transcriptional activation of human immunodeficiency virus type-1 (HIV-1) gene expression. HIV-1 encodes a transcriptional transactivator protein called Tat, which is expressed early in the viral life cycle and is absolutely required for viral replication and progression to disease. A regulatory element between +1 and +60 in the HIV-1 long terminal repeat which is capable of forming a stable stem-loop structure, designated TAR, is critical for Tat function. Tat interacts with cyclinT1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor b (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA. Recruitment of P-TEFb to TAR promotes transcription elongation. P-TEFb is a key enzyme in the control of transcription elongation by RNA polymerase II and there are two pools of P-TEFb, active and inactive, present in the cell. P-TEFb is inactivated by sequestration into a large ribonucleoprotein (RNP) complex containing the small nuclear RNA, 7SK, and the Hexim1 protein. Therefore, there are two RNP complexes, TAR-Tat-P-TEFb and 7SK-Hexim1-P-TEFb, which are important in HIV-1 gene expression, however, it is not known how the equilibrium between these two RNPs is modulated. We have developed innovative chemical and physical approaches to probe RNA-protein and protein-protein interactions. The proposed work addresses the structure, dynamics, and function of TAR-Tat-P-TEFb and 7SK-Hexim1-P-TEFb complexes by revealing the molecular network of RNA-RNA and RNA-protein interactions that govern assembly and stability of the RNP complexes.
Results of these studies would contribute to understanding the mechanisms of assembly and stability of RNA- protein complexes under physiological conditions and will improve our understanding of HIV-1 gene regulation by Tat. Since P-TEFb is a key enzyme regulating cellular gene expression, understanding the mechanism of P- TEFb activation would provide fundamental insight into a number of regulatory processes involved in the development of diseases such as cardiac hypertrophy, cancer, inflammation, and autoimmunity. These results would also be valuable in developing new strategies for blocking the expression of retroviral or other disease- related genes.
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