FOOTPRINTING WITH IRON(II)-GENERATED HYDROXYL RADICAL

提议的实验的主要目标是提供详细的 溶液中蛋白质-DNA复合体的结构信息。这个 利用铁(II)的化学方法进行实验的方法 过氧化氢产生羟基自由基。这是一个非常小的 活性分子与DNA的骨架发生反应，在DNA上留下缝隙 反应部位的链状结构。结合蛋白保护DNA不受 蛋白质-DNA接触点的裂解。这一观察形成了 最新开发的羟基自由基足迹法的基础 将在拟议的实验中使用。有三个具体目标 研究建议：1)利用DNA的X射线晶体结构-- 蛋白质复合体“校准”羟基自由基足迹法， 为了确定蛋白质-DNA复合体的结构特征 其中羟基自由基裂解反应敏感；2)使用 羟基自由基足迹法确定结构变化 发生在SV40的DNA复制启动过程中；以及3)研究 由多种蛋白质组成的“高阶”蛋白质-DNA复合体 绑定到DNA上。要研究的三个高阶系统是 噬菌体Lambda抑制子-操纵子系统，转录复合体 对于非洲爪哇的5S基因，包括TFIIIA、TFIIIB和TFIIIC，以及 果蝇Ubx蛋白与两种蛋白质的同时相互作用 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，提供了一种研究 活细胞遗传系统的组成部分。

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.