Metal Sites and Dynamics in Large RNA Molecules

描述（由申请人提供）：这项拟议研究的总体目标是研究大RNA分子中的特定金属结合位点及其对RNA功能的影响。RNA显示出丰富的细胞功能，涉及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，理解这种影响需要有效的直接探针用于金属位点群体和金属诱导的活性和构象变化。在本项目下进行的工作中，将研究Mg 2+和Mn 2+对金属结合和RNA结构的影响。RNA中金属配位环境的直接测量将使用Mn 2+作为EPR可检测的光谱探针进行。深入了解RNA构象变化将寻求使用位点特异性，EPR活性的氮氧自旋标记。两者合计，这些研究将提供重要的信息，RNA的结构作为溶液离子条件的函数，并将提供新的工具，更精细和构象动态的RNA复合物的调查。本提案的具体目标包括：1.研究I组内含子中独特的“金属离子核心”，目的是了解金属位点对该结构中折叠和稳定性的具体贡献2。确定的3-螺旋连接的延伸锤头状核酶，这将增加RNA折叠的基本性质的理解，也有助于在分离阳离子的折叠和催化在这种核酶的要求折叠的静电要求。3.确定金属离子在锤头状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参与了所有水平的基因调控，RNA的新作用仍在被发现。基于RNA的化学反应是最重要的细胞机器的中心，核糖体（蛋白质合成）和剪接体（基因剪接）。因此，了解RNA的基本性质以及它们如何受到环境的影响至关重要。

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.