Structure and Function of HIV Tat-TAR Complex

本研究的主要目的是了解参与人类免疫缺陷病毒1型（HIV- 1）基因表达转录激活的复杂组装体的结构和功能。HIV-1编码一种名为Tat的转录反激活蛋白，该蛋白在病毒生命周期的早期表达，是病毒复制和疾病进展所绝对需要的。在HIV-1长末端重复序列+1和+60之间的一个调控元件，能够形成一个稳定的茎环结构，称为TAR，对Tat功能至关重要。它与CDK9的调控伙伴cyclinTl （CycTl）相互作用

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.