Structure and Function of HIV Tat-TAR Complex

HIV Tat-TAR 复合物的结构和功能

• 批准号: 6932059
• 负责人: TARIQ M RANA
• 金额: $ 39.75万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932059
• 关键词: fluorescence resonance energy transfer; gene expression; genetic regulatory element; genetic transcription; high performance liquid chromatography; human immunodeficiency virus 1; intermolecular interaction; mass spectrometry; matrix assisted laser desorption ionization; protein protein interaction; protein structure function; structural biology; virus RNA; virus protein

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.

本研究的主要目标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件的结构和功能。 HIV-1 编码一种称为 Tat 的转录反式激活蛋白，该蛋白在病毒生命周期的早期表达，并且是病毒复制和疾病进展所必需的。 HIV-1长末端重复序列中+1和+60之间的调节元件能够形成稳定的茎环结构，称为TAR，对于Tat功能至关重要。 Tat 与 cyclinTl (CycTl) 相互作用，后者是 CDK9 的调节伙伴 正转录延伸因子 b (P-TEFb) 复合物，并与 CycT 1 协同结合 TAR RNA。 P-TEFb 募集至 TAR 可促进转录延伸。拟议的工作有三个具体目标。 具体目标 1：使用系统的位点特异性 RNA-蛋白质和蛋白质-蛋白质光交联来绘制 P-TEFb-Tat-TAR 复合物内 RNA-蛋白质和蛋白质-蛋白质相互作用的图谱。 具体目标 2：利用荧光共振能量转移来确定位点特异性引入 P-TEFb-Tat-TAR 复合物中的荧光探针对之间的距离。 具体目标 3：使用人工蛋白酶绘制三元 RNA-蛋白质复合物中蛋白质-蛋白质相互作用的图谱。 这些研究的结果将有助于理解生理条件下 Tat、P-TEFb 和 TAR RNA 之间相互作用的本质。了解 P-TEFb-Tat-TAR 复合物的结构和稳定性将极大地提高我们对这一复杂调节系统功能的理解。这些结果对于设计和合成 RNA-蛋白质和蛋白质-蛋白质相互作用的小分子抑制剂也很有价值。小分子对基因表达的选择性调控可能导致抗病毒和抗癌治疗药物的开发。