GLOBAL STRUCTURES OF RNA

为了实现其多样化的生物功能，RNA 必须折叠成特定的三级结构，从而为化学转化或蛋白质结合的识别特征创建活性位点。 实现生物活性所需的构象转变涉及螺旋、环和其他结构元件的大幅运动。 使用传统的结构分析方法很难研究这些动态构象转变。 时间分辨荧光共振能量转移 (tr-FRET) 是研究 RNA 构象转变的强大技术，因为它可以提供结构、热力学和动力学信息。 在本提案中，tr-FRET 和相关光谱方法将用于研究三个不同系统中生物学相关的 RNA 构象转变。 具体目标是： 1. 分析发夹核酶中三级结构的形成。 阐明核酶-底物复合物的底物结合和催化结构域有效对接的结构和热力学基础，并表征结构域对接过程中发生的碱基配对和堆积的变化。 2. 阐明离子和蛋白质诱导的 RNA 三向连接折叠的能量基础。 确定 16 S 和 5 S rRNA 三向连接的整体构象，并确定金属离子和蛋白质的结合如何与 RNA 内的构象变化相关联。 3. 确定来自 Rev Response Element 的大 RNA 片段的整体结构。 确定 HIV-1 Rev 和 Rev-Rev 多聚化在 RNA 上的结合是否会诱导螺旋重排。 这项研究的结果将揭示螺旋连接的结构如何指导远距离RNA结构域的对接并稳定具有生物活性的三级结构。 这些发现还将有助于了解蛋白质和金属离子辅助因子如何调节 RNA 的生物活性。 此外，这些结果将有助于设计改进的治疗性核酶，并有助于开发针对 RNA 靶点的药物。

To achieve its diverse range of biological functions, RNA must fold into specific tertiary structures that create active sites for chemical transformations or recognition features for protein binding. Conformational transitions necessary to achieve biological activity involve large movements of helices, loops and other structural elements. It is difficult to study these dynamic conformational transitions using conventional methods of structure analysis. Time-resolved fluorescence resonance energy transfer (tr-FRET) is a powerful technique for the study of RNA conformational transitions because it can provide structural, thermodynamic and kinetic information. In this proposal, tr-FRET and related spectroscopic methods will be used to study biologically-relevant RNA conformational transitions in three different systems. The specific aims are: 1. Analyze tertiary structure formation in the hairpin ribozyme. Elucidate the structural and thermodynamic basis for efficient docking of the substrate-binding and catalytic domains of the ribozyme-substrate complex and characterize changes in base pairing and stacking that occur during domain docking. 2. Elucidate the energetic basis for ion- and protein-induced folding of RNA three-way junctions. Determine the global conformations of three-way junctions from 16 S and 5 S rRNAs and establish how the binding of metal ions and proteins are linked to conformational changes within the RNA. 3. Determine the global structure of a large RNA fragment from the Rev Response Element. Establish whether binding of HIV-1 Rev and Rev-Rev multimerization on the RNA induce a rearrangement of helices. The results of this research will reveal how the structure of a helical junction can direct the docking of distant RNA domains and stabilize a biologically active tertiary structure. The findings will also contribute to an understanding of how proteins and metal ion cofactors can regulate the biological activity of RNA. In addition, the results will aid in the design of improved therapeutic ribozymes and contribute to the development of drugs directed against RNA targets.