TAT-TAR INTERACTIONS IN HIV PROBED BY CHEMICAL METHODS

通过化学方法探测 HIV 中的 TAT-TAR 相互作用

• 批准号: 3456486
• 负责人: TARIQ M RANA
• 金额: $ 12.07万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-15 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3456486
• 关键词: RNA binding protein; chelating agents; chemical cleavage; chemical structure; computer simulation; fluoresceins; genetic regulation; human immunodeficiency virus 1; intermolecular interaction; metal complex; nucleic acid sequence; oxidation reduction reaction; protein purification; rare earth element; virus RNA

The main focus of the proposed research is to understand the nature of interactions between the regulatory factor, Tat, and trans-acting responsive region of RNA (TAR) in human immunodeficiency virus-1 (HIV-1), and determine the structure of the Tat-TAR complex under physiological conditions. Knowledge of the structure of the protein-RNA complex, Tat-TAR, would greatly improve our understanding of the function of this complicated regulatory system. Structural information about the Tat-TAR complex will be obtained by using metal chelates site-specifically attached to RNA and peptides. Metal-catalyzed oxidative reactions will be initiated by the addition of redox-active metal salts and reducing agents in the presence of oxygen or hydrogen peroxide. These reactions cause the oxidative cleavage of RNA in the vicinity of the metal site. Chemical and enzymatic analyses will be carried out to locate the cleavage sites in RNA, and this information will reveal which bases in the folded RNA were close to the metal site. RNA labeled with metal chelates will be used to understand the three-dimensional folding of RNA in the presence and absence of the RNA-binding protein, since the protein binding causes a change in the tertiary structure of RNA and cleavage sites in the RNA sequence may differ in the presence or absence of RNA binding proteins. Interaction of protein side chains with RNA will be investigated by two methods: (a) RNA-binding peptides will be labeled site-specifically with metal chelates, and these peptides will be used to cleave RNA by metal-catalyzed oxidative reactions. Results from the analysis of the cleavage products will provide information about the protein contact sites on RNA. (b) Site-specifically labeled metal chelates can also serve as donors for fluorescence energy transfer experiments when lanthanide metal ions are used instead of redox active metals. Metal chelates which are incorporated into RNA site-specifically will be used as donors for energy transfer experiments. RNA-binding peptides will be labeled with fluorescein, acceptor molecules for energy transfer. The fraction of energy transfer will be used to determine the distance between peptide side chains and the metal site in RNA. The long term objectives of this project are to develop powerful and generally applicable methods to design RNA-binding peptides and metal complexes for RNA targeting. Computer modeling will be used to create the structure of TAR RNA compatible with the results of metal-catalyzed cleavage and fluorescence energy transfer experiments. On the basis of obtained structural data, small RNA-binding peptides will be synthesized, and these peptides will be used to deliver catalytic and efficient RNA-cleaving macrocyclic complexes to the target sites.

拟议研究的主要重点是了解 调节因子，TAT和反式作用之间的相互作用 人类免疫缺陷病毒1中RNA（焦油）的反应区域 （HIV-1），并确定tat-tar复合物的结构 生理条件。 了解结构 蛋白-rna复合物，tat-tar将大大提高我们的理解 这个复杂的监管系统的功能。 有关TAT-TAR复合体的结构信息将通过 使用特定于RNA和肽的金属螯合物特异性位点。 金属催化的氧化反应将通过加法开始 在存在的情况下， 氧或过氧化氢。这些反应引起氧化 金属部位附近RNA的切割。 化学和 将进行酶促分析以定位裂解位点 RNA，此信息将揭示折叠RNA中的哪些碱基 靠近金属遗址。 用金属螯合物标记的RNA将是 用于理解在存在下RNA的三维折叠 由于蛋白结合，RNA结合蛋白的缺失 导致RNA和切割位点的三级结构发生变化 RNA序列在存在或不存在RNA结合的情况下可能有所不同 蛋白质。 蛋白质侧链与RNA的相互作用将是 通过两种方法研究：（a）将标记RNA结合肽 特定于金属螯合物特定于现场，将使用这些肽 通过金属催化的氧化反应裂解RNA。 结果 分析产品的分析将提供有关 RNA上的蛋白质接触位点。 （b）特定于位置标记的金属 螯合物还可以用作荧光能量转移的供体 实验何时使用灯笼金属离子代替氧化还原活性 金属。 金属螯合物掺入RNA 特定地点将用作能量转移的捐助者 实验。 RNA结合肽将用荧光素标记 能量转移的受体分子。能量转移的比例 将用于确定肽侧链和 RNA中的金属位置。 该项目的长期目标是发展强大的 通常适用于设计RNA结合肽和金属的方法 RNA靶向的复合物。 计算机建模将用于创建 与金属催化的结果兼容的Tar RNA的结构 切割和荧光能量转移实验。 基于 在获得的结构数据中，小的RNA结合肽将是 合成的，这些肽将用于提供催化和 有效的RNA裂解大环复合物向目标部位。