FOOTPRINTING WITH IRON II GENERATED HYDROXYL RADICAL

为了表达其生物活性，DNA必须与 蛋白深入了解蛋白质与DNA的相互作用 将为疾病干预提供新的途径。的长期目标 这项工作是为了获得关于复杂的蛋白质-DNA的新信息， 存在于细胞中的复合物。新的研究方法已经被开发出来 DNA-蛋白质复合物的结构和能量学。这些方法 依赖于铁（H）EDTA与过氧化氢的化学反应， 在溶液中产生羟基自由基的方便方法。的 羟基通过夺取一个氢原子来切割DNA骨架 在DNA中留下一个单链缺口。这 化学可以用来制作非常高分辨率的“足迹”， 与DNA结合的蛋白质另一种基于羟基自由基化学的方法 是最近开发的缺失核苷实验， 直接信息的能量重要的接触， 与DNA结合位点的结合。本申请的具体目的 （1）将缺失核苷实验发展为定量研究， DNA中每个核苷酸相互作用的自由能的量度 与其同源蛋白的结合位点。(2)使用羟基自由基 足迹法和缺失核苷实验来表征DNA 一系列突变的同源结构域蛋白的结合，这些蛋白参与 指明了高等生物的发育途径。(3)使用化学 探针实验，包括缺失核苷实验， 确定RNA聚合酶-DNA复合物的结构特征， 在转录起始和延伸中。(4)开发一种新的方法 用于制作活体DNA-蛋白质复合物的高分辨率足迹， 细胞，使用伽马射线在体内产生羟基自由基。这个新 将应用体内足迹法来表征组织- 生长激素基因的特异性表达。

For its biological activity to be expressed, DNA must be associated with protein. Deeper understanding of the interactions of proteins with DNA will provide new avenues for disease intervention. The long term goal of this work is to gain new information on the complicated protein-DNA complexes that exist in the cell. New methods have been developed to study the structure and energetics of DNA-protein complexes. These methods depend on the chemistry of iron(H) EDTA with hydrogen peroxide as a convenient means of generating the hydroxyl radical in solution. The hydroxyl radical cleaves the DNA backbone by abstracting a hydrogen atom from a deoxyribose, leaving a single-stranded gap in the DNA. This chemistry can be used to make very high resolution "footprints" of proteins bound to DNA. Another method based on hydroxyl radical chemistry is the recently-developed Missing Nucleoside Experiment, which provides direct information on the energetically important contacted that a protein makes with its DNA binding site. The Specific Aims of this application are: (1) to develop the Missing Nucleoside Experiment into a quantitative measure of the free energy of interaction of each nucleotide in a DNA binding site with its cognate protein. (2) to use hydroxyl radical footprinting and the Missing Nucleoside Experiment to characterize the DNA binding of a series of mutated homeodomain proteins, which are involved in specifying developmental pathways in higher organisms. (3) to use chemical probe experiments, including the Missing Nucleoside Experiment, to determine the structural features of RNA polymerase-DNA complexes engaged in transcription initiation and elongation. (4) to develop a new method for making high resolution footprints of DNA-protein complexes in living cells, using gamma radiation to produce hydroxyl radical in vivo. This new in vivo footprinting methods will be applied to characterizing the tissue- specific expression of the growth hormone gene.