ROLE OF TAT IN HIV TRANSCRIPTION INITIATION

描述：（改编自申请人摘要） 本项目旨在阐明达特的分子机制 转录起始期间的反式激活。 离散局部化 在转录起始中有活性的达特功能结构域将被 通过定点诱变和评价达特突变体确定 HIV在启动前和启动的不同阶段的转录以及 通过构建GAL 4/达特融合蛋白并对其进行评价， 体外和体内对HIV转录影响。 解剖 达特增强预引发复合物形成的机制 HIV转录将通过确定结合和 使用凝胶位移的预引发复合物组装的解离速率 和足迹分析，通过测定Tat-蛋白质相互作用 使用共免疫沉淀分析、共沉降分析、蛋白质 过滤器结合、达特亲和层析和达特测定 对前起始复合物组分的功能活性的影响。 Tat介导的对合成步骤的影响的测定 转录起始将通过定量以下物质的丰度来提供： 在转录起始时合成的第一个二核苷酸， 生产前引发复合物。

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.