FOOTPRINTING WITH IRON(II)-GENERATED HYDROXYL RADICAL

铁 (II) 生成的羟基自由基的足迹

• 批准号: 3300424
• 负责人: THOMAS D TULLIUS
• 金额: $ 16.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3300424
• 关键词: DNA binding protein; DNA footprinting; Drosophilidae; X ray crystallography; Xenopus; adenosine triphosphate; bacteriophage lambda; ethylenediaminetetraacetate; hydrogen peroxide; hydroxyl radical; simian virus 40

The main objective of the proposed experiments is to provide detailed structural information on protein-DNA complexes in solution. The experimental method to be used make use of chemistry of iron (II) with hydrogen peroxide to generate the hydroxyl radical. This very small and reactive molecule reacts with the backbone of DNA, leaving gaps in the DNA strand at the site of reaction. Bound protein protects the DNA from cleavage at the points of protein-DNA contact. This observation forms the basis of the newly-developed hydroxy radical "footprinting" method that will be used in the proposed experiments. There are three specific goals of the research proposed: 1) to use X-ray crystal structures of DNA- protein complexes to "calibrate" the hydroxyl radical footprinting method, in order to determine the structural features of protein-DNA complexes to which the hydroxyl radical cleavage reaction is sensitive; 2) to use the hydroxyl radical footprinting method to determine structural changes that occur during initiation of DNA replication in SV40; and 3) to study "higher-order" protein-DNA complexes that are made up of multiple proteins bound to DNA. The three higher-order systems to be studied are the bacteriophage lambda repressor-operator system, the transcription complex for the 5S genes of Xenopus that includes TFIIIA, TFIIIB and TFIIIC, and the simultaneous interaction of the UBX protein of Drosophila with two binding sites near its own gene. The ability of hydroxyl radical footprinting to determine structural details for complicated protein-DNA systems, as exemplified in goal 3), provides a new way to study the components of the genetic systems of the living cell.

拟议实验的主要目的是提供详细的 溶液中蛋白质-DNA复合物的结构信息。 这 使用的实验方法利用铁（II）的化学方法 过氧化氢以产生羟基自由基。 这个很小， 反应性分子与DNA的主链反应，在DNA中留下间隙 反应部位的链。 结合蛋白可以保护DNA免受 蛋白-DNA接触点的切割。 这个观察构成了 新开发的羟基激进的“足迹”方法的基础 将用于拟议的实验。 有三个特定目标 提出的研究：1）使用DNA-的X射线晶体结构 蛋白质复合物“校准”羟基自由基足迹法， 为了确定蛋白-DNA复合物的结构特征 羟基自由基裂解反应是敏感的； 2）使用 羟基自由基足迹法确定结构性变化 在SV40中启动DNA复制期间发生； 3）学习 由多种蛋白质组成的“高阶”蛋白-DNA复合物 与DNA结合。 要研究的三个高阶系统是 噬菌体lambda阻遏物系统，转录复合物 对于包括TFIIIA，TFIIIB和TFIIIC的5S基因，以及 果蝇的UBX蛋白与两个的同时相互作用 绑定位点附近的基因。 羟基的能力 足迹以确定复杂蛋白DNA的结构细节 在目标3中的例证系统中，系统提供了一种研究的新方法 活细胞的遗传系统的成分。