Metal Sites and Dynamics in Large RNA Molecules

描述(申请人提供)：拟议研究的总体目标是调查大RNA分子中的金属结合部位及其对RNA结构、稳定性和动力学的影响。RNA通过与其他核酸和蛋白质的复杂相互作用显示出丰富的细胞功能。RNA-金属相互作用对RNA的结构和功能都是至关重要的，需要有效、直接地探测金属位置对RNA结构生物学的影响。在该项目下开展的工作中，将使用Mn2+作为EPR可检测的光谱探针来测量RNA系统中金属结合的直接测量。研究了镁、锰离子对金属结合、RNA结构和热力学参数的影响。对RNA构象动力学的洞察将使用位点特定的、EPR活性的氮氧化物自旋标记来寻求。综上所述，这些研究将提供关于RNA结构和稳定性随溶液条件的变化的重要信息，并将为更精细和构象动态的RNA络合物的研究提供新的工具。当前提议的具体目标包括：研究来自I族内含子的独特的‘金属离子核’，目的是具体了解金属离子如何促进该结构基序中的折叠和稳定性。确定金属离子亲和力以及金属对发夹状核酶中折叠和催化的影响。开发针对位点的EPR活性自旋标记来研究RNA中的动力学和距离，应用于锤头蛋白和第一组内含子P4-P6亚区中金属依赖的折叠途径。在剪接体活性部位的U2-U6 RNA模型体系中，研究金属离子对折叠和活性的影响。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to investigate metal binding sites in large RNA molecules and their influence on RNA structure, stability, and dynamics. RNA displays a rich array of cellular functions through complex interactions with other nucleic acids and proteins. RNA-metal interactions are critical to both structure and function, and effective, direct probes of the influence of metal sites on the structural biology of RNA are required. In work carried out under this project, direct measurements of metal-binding in RNA systems will be measured using Mn2+ as an EPR-detectable spectroscopic probe. The influence of both Mg2+ and Mn2+ on metal binding, RNA structure, and thermodynamic parameters will be investigated. Insight into the conformational dynamics of RNA will be sought using site-specific, EPR-active nitroxide spin labels. Taken together, these studies will provide important information about RNA structure and stability as a function of solution conditions, and will provide new tools for the investigation of more elaborate and conformationally dynamic RNA complexes.Specific aims of the current proposal include: Investigating the unique 'metal ion core' from the Group I Intron, with a goal of understanding specifically how metal ions contribute to folding and stability in this structural motif. Determining metal ion affinities and influence of metals on folding and catalysis in the hairpin ribozyme. Developing methods for site-specific EPR-active spin labels to study dynamics and distances in RNA, with application to metal-dependent folding pathways in the hammerhead and Group I Intron P4-P6 subdomain. Investigating the influence of metal ions on folding and activity in a U2-U6 RNA model system for the active site of the spliceosome.