FOOTPRINTING WITH IRON(II)-GENERATED HYDROXYL RADICAL

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 的 使用的实验方法利用铁（II）的化学性质， 过氧化氢以产生羟基自由基。 这个非常小的， 反应分子与DNA的骨架反应，在DNA中留下缺口 反应位点的链。 结合蛋白质保护DNA 蛋白质-DNA接触点处的切割。 这一观察构成了 根据新开发的羟基自由基“足迹”方法， 将用于拟议的实验。 具体目标有三个 该研究提出：1）使用DNA的X射线晶体结构- 蛋白质复合物来“校准”羟基自由基足迹法， 为了确定蛋白质-DNA复合物的结构特征， 其中羟基自由基裂解反应敏感; 2）使用 羟基自由基足迹法，以确定结构变化， 在SV 40中DNA复制启动期间发生;和3）研究 由多种蛋白质组成的“高级”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λ阻遏子-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 在自身基因附近的结合位点 羟自由基能力 足迹法用于确定复杂蛋白质-DNA的结构细节 如目标3所示，提供了一种新的研究方法， 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.