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.

所提出的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 所使用的实验方法利用铁（II）的化学 过氧化氢产生羟基自由基。 这个非常小而且 反应分子与 DNA 骨架发生反应，在 DNA 中留下间隙 链位于反应位点。 结合蛋白可保护 DNA 免受 蛋白质-DNA 接触点处的裂解。 这一观察形成了 新开发的羟基自由基“足迹”方法的基础 将在建议的实验中使用。 具体目标有3个 研究建议：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， 以确定蛋白质-DNA复合物的结构特征 对羟基自由基裂解反应敏感； 2）使用 羟基自由基足迹法测定结构变化 发生在 SV40 DNA 复制起始期间； 3）学习 由多种蛋白质组成的“高阶”蛋白质-DNA 复合物 与 DNA 结合。 要研究的三个高阶系统是 噬菌体 lambda 阻遏物操纵子系统，转录复合物 对于非洲爪蟾的 5S 基因，包括 TFIIIA、TFIIIB 和 TFIIIC，以及 果蝇的 UBX 蛋白同时与两个 其自身基因附近的结合位点。 羟基自由基的能力 足迹法确定复杂蛋白质-DNA 的结构细节 系统，如目标 3）所示，提供了一种新的方法来研究 活细胞遗传系统的组成部分。