Metal Sites and Dynamics in Large RNA Molecules

描述（由申请人提供）：拟议研究的总体目标是研究大RNA分子中的金属结合位点及其对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的结构和功能都至关重要，因此需要金属位点对RNA结构生物学影响的有效、直接的探针。在该项目下进行的工作中，将使用Mn 2+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。将研究Mg 2+和Mn 2+对金属结合、RNA结构和热力学参数的影响。深入了解RNA的构象动力学将寻求使用位点特异性，EPR活性的氮氧自旋标记。总之，这些研究将提供关于RNA结构和稳定性作为溶液条件的函数的重要信息，并将为研究更精细和构象动态的RNA复合物提供新的工具。当前提案的具体目标包括： 研究I组内含子中独特的“金属离子核心”，目的是具体了解金属离子如何有助于该结构基序的折叠和稳定性。 发夹状核酶中金属离子亲和性的测定及其对折叠和催化的影响。 开发用于位点特异性EPR活性自旋标记的方法，以研究RNA中的动力学和距离，并应用于锤头和I组内含子P4-P6亚结构域中的金属依赖性折叠途径。 研究金属离子对剪接体活性位点的U2-U6 RNA模型系统中折叠和活性的影响。

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.