Metal Sites and Dynamics in Large RNA Molecules

描述（由申请人提供）：本研究的总体目标是研究大RNA分子中特定的金属结合位点及其对RNA功能的影响。RNA显示了丰富的细胞功能，包括与其他核酸和蛋白质的复杂相互作用。rna -金属相互作用对结构和功能都至关重要，理解这种影响需要对金属位点数量、金属诱导的活性和构象变化进行有效、直接的探测。在本项目开展的工作中，将研究Mg2+和Mn2+对金属结合和RNA结构的影响。直接测量RNA中的金属配位环境将使用Mn2+作为EPR可探测的光谱探针进行。洞察RNA构象变化将寻求使用位点特异性，epr活性氮氧化物自旋标签。综上所述，这些研究将提供RNA结构作为溶液离子条件的功能的重要信息，并将为研究更复杂和构象动态的RNA复合物提供新的工具。当前提案的具体目标包括：1。研究来自I组内含子的独特“金属离子核”，目的是了解金属位点对该结构的折叠和稳定性的具体贡献2。确定扩展锤头核酶中3-螺旋结折叠的静电要求，这将增加对RNA折叠基本特性的理解，也有助于分离该核酶折叠和催化的阳离子要求。3. 确定金属离子在锤头核酶切割反应中的作用，使用靶向光谱方法精确定义金属- rna相互作用。核糖核酸（RNA）可以说是细胞中最重要的分子。RNA参与调控基因的各个层面，RNA的新作用还在不断被发现。基于rna的化学反应是最重要的细胞机器，核糖体（蛋白质合成）和剪接体（基因剪接）的中心。因此，了解RNA的基本特性以及它们如何受到环境的影响是至关重要的。

DESCRIPTION (provided by applicant): The overall goal of this proposed research is to investigate specific metal binding sites in large RNA molecules and their influence on RNA function. RNA displays a rich array of cellular functions involving complex interactions with other nucleic acids and proteins. RNA-metal interactions are critical to both structure and function, and understanding this influence requires effective, direct probes for metal site population and metal-induced activity and conformational changes. In work carried out under this project, the influence of both Mg2+ and Mn2+ on metal-binding and RNA structure will be investigated. Direct measurements of the metal coordination environment in RNA will be performed using Mn2+ as an EPR- detectable spectroscopic probe. Insight into RNA conformational changes will be sought using site-specific, EPR-active nitroxide spin labels. Taken together, these studies will provide important information about RNA structure as a function of solution ionic conditions, and will provide new tools for the investigation of more elaborate and conformationally dynamic RNA complexes. Specific aims of the current proposal include: 1. Investigating the unique 'metal ion core' from the Group I Intron, with a goal of understanding the specific contributions of metal sites to folding and stability in this structure 2. Determining the electrostatic requirements for folding of a 3-helix junction in the extended Hammerhead ribozyme, which will increase understanding of fundamental properties of RNA folding and also aid in separating cation requirements for folding and for catalysis in this ribozyme. 3. Determining the roles of metal ions in the Hammerhead ribozyme cleavage reaction, using targeted spectroscopic methods to define precisely metal-RNA interactions. Ribonucleic acid (RNA) is arguably the most important molecule in the cell. RNA is involved in regulating genes at all levels, and new roles for RNA are still being discovered. RNA-based chemical reactions are at the center of the most important cellular machines, the ribosome (protein synthesis) and spliceosome (gene splicing). It is therefore critical to understand the basic properties of RNA and how they are influenced by the environment.