Structure and Function of HIV Tat-TAR Complex

这项研究的主要目的是了解参与人类免疫缺陷病毒1型(HIV-1)基因转录激活的复杂组件的结构和功能。HIV-1编码一种名为TAT的转录反式激活蛋白，它在病毒生命周期的早期表达，是病毒复制和疾病发展所必需的。HIV-1长末端重复序列中+1到+60之间的调节元件能够形成稳定的茎环结构，命名为TAR，对TAT功能至关重要。TAT与CDK9的监管合作伙伴细胞周期蛋白T1(CycTl)在 阳性转录延伸因子b(P-TEFb)复合体，并与CycT 1协同结合到TAR RNA上。将P-TEFb招募到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复合体中RNA-蛋白质和蛋白质-蛋白质的相互作用图。 具体目标2：利用荧光共振能量转移来确定定点引入P-TEFb-TAT-TAR络合物的荧光探针对之间的距离。 具体目的3：使用人工蛋白酶绘制三元RNA-蛋白质复合体中蛋白质-蛋白质相互作用的图谱。 这些研究结果将有助于理解生理条件下TAT、P-TEFb和TAR RNA之间相互作用的性质。了解P-TEFb-TAT-TAR复合体的结构和稳定性将极大地提高我们对这一复杂调控系统功能的理解。这些结果也将对设计和合成RNA-蛋白质和蛋白质-蛋白质相互作用的小分子抑制剂具有重要意义。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.