ROLE OF TAT IN HIV TRANSCRIPTION INITIATION

描述：(改编自申请人的摘要)总体目标 本课题旨在阐明Tat的分子机制。 转录启动过程中的反式激活。离散化的局部化 在转录起始中活跃的TAT功能结构域将是 Tat突变体的定点诱变及鉴定 HIV在启动前和启动前不同阶段的转录和 GAL4/TAT融合蛋白的构建及鉴定 对HIV体外和体内转录的影响。对中国传统文化的剖析 TAT促进前引发复合体形成的机制(S) HIV转录将通过确定关联性和 凝胶位移法研究预引发络合物组装的解离速率 和足迹分析，通过确定TAT-蛋白质相互作用 使用免疫共沉淀分析，共沉淀分析，蛋白质 滤膜结合、TAT亲和层析和TAT的测定 对预引发络合物组分功能活性的影响。 TAT介导的合成步骤效应的测定 转录的启动将通过量化的丰度提供 在转录起始时合成的第一个二核苷酸 生产性预引发复合体。

DESCRIPTION: (Adapted from applicant's abstract) The general objective of this project is to elucidate the molecular mechanisms of Tat transactivation during transcription initiation. Localization of discrete Tat functional domains active in transcription initiation will be determined by site-directed mutagenesis and evaluation of Tat mutants on HIV transcription at different stages of pre-initiation and initiation and by construction and evaluation of GAL4/Tat fusion proteins for their effect on HIV transcription in vitro and in vivo. Dissection of the mechanism(s) by which Tat enhances pre-initiation complex formation during HIV transcription will be accomplished by determination of association and disassociation rates of pre-initiation complex assembly using gel shift and footprinting assays, by determination of Tat-protein interactions using co-immunoprecipitation assays, co-sedimentation analysis, protein filter binding, Tat affinity chromatography and by determination of Tat effects on functional activities of pre-initiation complex components. Determination of Tat-mediated effects on the synthetic step of transcription initiation will be afforded by quantitating the abundance of the first dinucleotide synthesized upon transcription initiation from productive pre-initiation complexes.