METAL SITES AND DYNAMICS IN LARGE RNA MOLECULES

描述：（改编自申请人的摘要）长期目标 这项研究计划的目的是帮助理解结构， 并在RNA分子中发挥作用， 交互. RNA在控制遗传信息中的重要性， 我们在基因治疗应用中的潜力，推动了相当大的电流 对RNA结构的物理和化学基础感兴趣， 化学反应性 金属-RNA相互作用在稳定 特定的RNA三级结构，并参与RNA催化的 化学反应。 RNA中金属位点的性质对于 结构和反应性之间的关系，以及增加知识 金属-RNA相互作用的研究可能有助于设计更有效的RNA 催化剂，例如用作体内治疗剂。 该计划的一个新的重点是使用光谱技术， 获得关于催化RNA中金属配位位点的特定信息 分子或核酶。 长期以来，对蛋白质中金属的研究 密集使用基于金属的光谱方法， 关于结合位点的数量和类型的信息，以及 特定金属配体的鉴定。 对于RNA分子，最近 大规模样品制备的进展， 来自生化实验的知识和来自X射线的有趣预测 晶体学研究使得这种光谱学研究既可行， 很及时 这项工作的具体目标涉及大规模测量金属-RNA相互作用 组I内含子形式嗜热四膜虫。 这种核酶催化 金属依赖的自剪接反应，可以分为两个稳定的 子域，并提供了一个相对良好的研究模型系统， 预期具有多种不同的金属离子位点。 数量和 I组内含子中二价阳离子结合位点的亲和力及其 子域，金属离子特异性，和配体环境将被追求 使用光谱技术，包括EPR、NMR、电子-核双 共振光谱学和其他需要的。 EPR活性自旋标签将 可以用来探测这个大RNA系统中的运动动力学， 绘制出金属位点和三级相互作用。 除了获得 关于I组内含子的具体信息，这些研究通常 阐明了大RNA分子以前未知的性质。

DESCRIPTION: (adapted from applicant's abstract) The long term objectives of this research program are to contribute to the understanding of structure and function in RNA molecules by determining principles of metal-RNA interactions. The importance of RNA in controlling genetic information, and potential for us in Gene therapy applications, drive considerable current interest in the physical and chemical foundations of RNA structure and chemical reactivity. Metal-RNA interactions are critical in stabilizing specific RNA tertiary structures, and in participating in RNA-catalyzed chemical reactions. The properties of metal sites in RNA are fundamental to the relationship between structure and reactivity, and increased knowledge of metal-RNA interactions ma aid in the design of more efficient RNA catalysts, for example to be used as therapeutic agents in vivo. A novel emphasis of this program concerns using spectroscopic techniques to obtain specific information about metal coordination sites in catalytic RNA molecules, or ribozymes. The study of metals in proteins has long made intensive use of metal-based spectroscopic methods, which provide information about numbers and types of binding sites as well as identification of specific metal ligands. For RNA molecules, recent advances in large-scale sample preparation, an ever-increasing body of knowledge from biochemical experiments and intriguing predictions from X-ray crystallography studies make such spectroscopic studies both feasible and timely. Specific aims of this work involve measuring metal-RNA interactions in large Group I intron form Tetrahymena thermophilia. This ribozyme catalyzes a metal-dependent self-splicing reaction, can be separated into two stable subdomains, and provides a relatively well-studied model system that is expected to have a variety of different metal ion sites. The number and affinity of divalent cation binding sites in the group I intron and its subdomains, metal ion specificity's, and ligand environments will be pursued using spectroscopic techniques including EPR, NMR, electron-nuclear double resonance spectroscopy, and others as needed. EPR-active spin labels will be used to probe the dynamics of motions in this large RNA system, and to map out metal sites and tertiary interactions. In addition to gaining specific information about the group I intron, these studies will in general elucidate previously unknown properties of large RNA molecules.