MECHANISMS OF HIV1 TAT TRANSACTIVATION

描述：拟议研究的总体目标是得出一个 对一种小病毒蛋白TAT如何控制病毒的生物化学理解 艾滋病毒基因组的表达。这项研究的重点将是自行车 依赖激酶，P-TEFb，是控制伸长的关键因素，通过 RNA聚合酶II，是TAT反式激活所必需的。 HIV-1Tat与PTEFb相互作用，增强其在HIV-LTR中的功能。 首先，人类P-TEFb将通过确定其亚基来表征 结构。Pitalre、激酶亚基和几种可能的周期蛋白 将对亚基进行分析，以确定它们是否与 活性P-TEFb。下一步，高效交互的要求 P-TEFb与TAT的结合将使用体外结合研究进行检测 固定化TAT和P-TEFb复合体及其体内免疫沉淀 实验。对蛋白质或RNA辅因子的潜在需求将 被称呼为。最后，进行了一系列实验，以解决生化问题 TAT介导增强P-TEFb作用的机制将是 已执行。其中将包括TAT的详细优化 使用HeLa和Jurkat细胞提取物的体外反式激活。 TAT对P-TEFb、F2、SPT4/SPT5相关性的影响 复合体，以及其他与转录复合体的因子将 下定决心。HIV-LTR似乎特别敏感的原因 TO PTEFb抑制剂也将被探索。此外，酵母菌系统 将被设计成可以允许研究TAT反式激活和 P-TEFb与TAT在体内的相互作用艾滋病毒的流行正在日益严重。 国家和国际关注的和最有效的 治疗是指将不同药物组合使用的治疗方法。 了解Tat反式激活可能会导致新药的发现 与艾滋病作斗争。

DESCRIPTION: The overall goal of the proposed research is to come to a biochemical understanding of how a small viral protein, Tat, controls the expression of the HIV genome. The focus of the study will be on a cyclin dependent kinase, P-TEFb, that is a key factor controlling elongation by RNA polymerase II and is specifically required for Tat transactivation. HIV-1 Tat interacts with PTEFb and enhances its function at the HIV-LTR. First, human P-TEFb will be characterized by determining its subunit structure. PITALRE, the kinase subunit, and several putative cyclin subunits will be analyzed to determine if they are in complexes with active P-TEFb. Next, the requirements for efficient interaction between P-TEFb with Tat will be examined using in vitro binding studies with immobilized Tat and P-TEFb complexes and in vivo immunoprecipitation experiments. The potential requirement for protein or RNA cofactors will be addressed. Finally, a series of experiments to address the biochemical mechanism of Tat mediated enhancement of the action of P-TEFb will be performed. These will include a detailed optimization of Tat transactivation in vitro using extracts from both HeLa and Jurkat cells. The effect of Tat on the association of P-TEFb, factor 2, the SPT4/SPT5 complex, and other factors with the transcription complex will be determined. The reason that the HIV-LTR seems to be especially sensitive to PTEFb inhibitors will also be explored. In addition, a yeast system will be designed that may allow the study of Tat transactivation and the interaction of P-TEFb and Tat in vivo. The HIV epidemic is a growing national as well as international concern and the most effective treatments are those incorporating a combinatorial use of different drugs. Understanding Tat transactivation may lead to the discovery of new drugs to battle AIDS.