MECHANISMS OF HIV1 TAT TRANSACTIVATION

描述：拟议研究的总体目标是 一个小的病毒蛋白，达特，如何控制生物化学的理解， HIV基因组的表达。 这项研究的重点将是细胞周期蛋白 依赖性激酶，P-TEFb，其是控制延伸的关键因子， RNA聚合酶II，并且是达特反式激活所特异性需要的。 HIV-1达特与PTEFb相互作用并增强其在HIV-LTR的功能。 首先，人P-TEFb将通过确定其亚基来表征 结构PITALRE、激酶亚基和几种假定的细胞周期蛋白 将分析亚基以确定它们是否与 活性P-TEFb。 其次，在以下方面进行有效交互的要求 将使用体外结合研究检查P-TEFb与达特的结合， 固定化达特和P-TEFb复合物和体内免疫沉淀 实验 对蛋白质或RNA辅因子的潜在需求将 被解决。 最后，一系列的实验，以解决生化 达特介导的P-TEFb作用增强的机制将是 执行。 这些将包括对达特的详细优化 使用来自HeLa和Jurkat细胞的提取物进行体外反式激活。 达特对P-TEFb、因子2、SPT 4/SPT 5的缔合的影响 复合物，和其他因子与转录复合物将是 测定 HIV-LTR似乎特别敏感 PTEFb抑制剂也将被探索。 此外，酵母系统 将被设计为可以允许研究达特反式激活和 P-TEFb和达特在体内相互作用。 艾滋病毒的流行是一个日益增长的 国家和国际关注的问题， 治疗是结合不同药物的组合使用的治疗。 了解达特反式激活可能会导致发现新的药物 来对抗艾滋病

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.