METAL SITES AND DYNAMICS IN LARGE RNA MOLECULES

描述：(改编自申请者摘要)长期目标 这项研究计划的目的是为了帮助理解结构 并通过确定金属-RNA的原理在RNA分子中发挥作用 互动。RNA在控制遗传信息方面的重要性，以及 我们在基因治疗应用方面的潜力，推动了相当大的电流 对RNA结构和结构的物理和化学基础感兴趣 化学反应。金属-核糖核酸相互作用是稳定的关键 特定的RNA三级结构，并在参与RNA催化 化学反应。核糖核酸中金属位点的性质是 结构和反应性之间的关系，以及知识的增长 金属-RNA相互作用有助于设计更有效的RNA 催化剂，例如用作体内的治疗剂。 这个项目的一个新的重点是使用光谱技术来 获取催化RNA中金属配位的特定信息 分子，或核酶。对蛋白质中金属的研究由来已久 密集使用基于金属的光谱方法，这提供了 有关结合位点的数量和类型以及 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，一个不断增长的主体 来自生化实验的知识和来自X射线的有趣预测 结晶学研究使这种光谱研究既可行又 太及时了。 这项工作的具体目标涉及在大范围内测量金属-RNA相互作用 I组内含子形成嗜热四膜虫。这种核酶催化一种 金属依赖的自剪接反应，可以分为两个稳定的 子域，并提供了一个相对研究较好的模型系统，即 预计会有各种不同的金属离子位置。数字和 I族内含子二价阳离子结合位点及其亲和力的研究 亚域、金属离子专一性和配基环境将被研究 使用包括EPR、核磁共振、电子-核双谱在内的光谱技术 共振光谱学，以及根据需要进行其他测试。EPR-活动自旋标签将 用于探测这个大的RNA系统中的运动动力学，并 绘制出金属位置和第三级相互作用图。除了获得 关于第一组内含子的具体信息，这些研究将大体上 阐明以前不为人知的大RNA分子的性质。

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.