The main goal of the proposed research is to understand the architecture 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 cyclinTl (CycTl), a regulatory partner of CDK9 in the positive transcription elongation factor b (P-TEFb) complex, and binds cooperatively with CycT 1 to TAR RNA. Recruitment of P-TEFb to TAR promotes transcription elongation. The proposed work has three specific aims.
Specific aim 1 : To use systematic site-specific RNA-protein and protein-protein photocrosslinking to map RNA-protein and protein-protein interactions within the P-TEFb-Tat-TAR complex.
Specific aim 2 : To use fluorescence resonance energy transfer to define distances between pairs of fluorescent probes site-specifically introduced into the P-TEFb-Tat-TAR complex.
Specific aim 3 : To use artificial proteases to map protein-protein interactions in the ternary RNA-protein complex. Results of these studies would contribute to understanding the nature of interactions between Tat, P-TEFb, and TAR RNA under physiological conditions. Knowledge of the architecture and stability of the P-TEFb-Tat-TAR complex would greatly improve our understanding of the function of this complicated regulatory system. These results would also be valuable in designing and synthesis of small molecule inhibitors of RNA-protein and protein-protein interactions. Selective regulation of gene expression by small molecules could lead to the development of antiviral and anticancer therapeutic agents.
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