X-RAY DIFFRACTION ANALYSIS OF DNA BINDING PROTEINS

这项研究将建立精确定义的蛋白质/核酸结构 系统通过X射线结晶学技术的应用 目的是为描述提供可靠的物理基础 蛋白质-核酸相互作用。这将有助于分子生物学家 在原子水平上推断，遗传信息是 调节并转化为特定的细胞事件。此外，它还将 有助于我们理解 染色体的结构。通过直接可视化关键的 相互作用，这些大分子的结构元素负责 它们的生物活性可以明确定义。 这项研究将围绕三维结构的解决方案和高 Fd噬菌体基因5蛋白的精确提纯 牛肉胰腺脱氧核糖核酸酶。此外，这些蛋白质的复合体 用差分傅立叶和分子分析方法分析寡聚脱氧核苷酸 替换技术来绘制关键的原子相互作用图。突变形式的 来自密切相关病毒的基因5蛋白和同源蛋白将是 通过结晶学分析得出这些分子细节 对修饰特别敏感 还将尝试分离、纯化和结晶DNA结合 来自其他生物的蛋白质，目的是扩大 可用于单晶X射线衍射的结构和相互作用 分析。在这方面的初步努力将针对DNA 解开T4噬菌体(基因32蛋白)、大肠杆菌和小牛的蛋白 胸腺，培养的人成纤维细胞的DNA结合蛋白和乳糖 来自大肠杆菌的操纵子抑制子。

This research will establish precisely defined protein/nucleic acid structural systems though the application of X-ray crystallographic techniques for the purpose of providing a sound physical basis for the description of protein-nucleic acid interactions. This will assist molecular biologists in deducing, at an atomic level, the means by which genetic information is regulated and transformed into specific cellular events. It furthermore will contribute to our understanding of the fundamental principles underlying the structure of the chromosome. By direct visualization of the crucial interactions, the structural elements of these macromolecules responsible for their biological activity can be explicitly defined. This research will encompass the three-dimensional structure solution and high precision refinement of the Gene 5 protein from fd bacteriophage and deoxyribonuclease from beef pancreas. In addition, complexes of these proteins with oligo-deoxynucleotides will be analyzed by difference Fourier and molecular replacement techniques to map the crucial atomic interactions. Mutant forms of the Gene 5 protein and homologous proteins from closely related viruses will be analyzed by crystallographic analysis to derive those molecular details particularly sensitive to modification Attempts will also be made to isolate, purify and crystallize DNA binding proteins from other organisms for the purpose of expanding the range of structures and interactions available for single crystal X-ray diffraction analyses. The initial efforts in this regard will be directed toward the DNA unwinding proteins from T4 bacteriophage (the Gene 32 protein), E. coli and calf thymus, the DNA binding protein from cultured human fibroblasts and the lactose operon repressor from E. coli.