Structure and Function of HIV Tat-TAR Complex

拟议的研究的主要目标是了解的架构和功能的复杂组件参与人类免疫缺陷病毒1型（HIV- 1）基因表达的转录激活。HIV-1编码一种称为达特的转录反式激活蛋白，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，对达特功能至关重要。达特与细胞周期蛋白T1（CycT 1）相互作用，细胞周期蛋白T1是CDK 9在细胞周期中的调节伙伴。 正转录延伸因子B（P-TEF B）复合物，并与CycT 1协同结合至TAR RNA。P-TEFb向TAR的募集促进转录延伸。拟议的工作有三个具体目标。 具体目标1：使用系统性位点特异性RNA-蛋白质和蛋白质-蛋白质光交联来绘制P-TEFb-Tat-TAR复合物内的RNA-蛋白质和蛋白质-蛋白质相互作用。 具体目标2：使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-蛋白质复合物中的蛋白质-蛋白质相互作用。 这些研究的结果将有助于理解生理条件下达特，P-TEFb和TAR RNA之间的相互作用的性质。了解P-TEFb-Tat-TAR复合物的结构和稳定性将大大提高我们对这一复杂调控系统功能的理解。这些结果也将是有价值的设计和合成的小分子抑制剂的RNA-蛋白质和蛋白质-蛋白质相互作用。通过小分子选择性调节基因表达可能导致抗病毒和抗癌治疗剂的发展。

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.