GLOBAL STRUCTURES OF RNA

为了实现其不同的生物学功能，RNA必须折叠成特定的三级结构，为化学转化创造活性部位，或为蛋白质结合创造识别特征。实现生物活性所需的构象转变涉及螺旋、环和其他结构元素的大量移动。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技术能够提供结构、热力学和动力学信息，是研究RNA构象转变的一种强有力的技术手段。在这项建议中，TRRET和相关的光谱方法将被用来研究三个不同系统中与生物相关的RNA构象转变。具体目的是：1.分析发夹状核酶的三级结构形成。阐明核酶-底物复合体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.阐明了离子和蛋白质诱导的RNA三向连接折叠的能量基础。从16个S和5个S的RNA中确定三向连接的全球构象，并建立金属离子和蛋白质的结合如何与RNA中的构象变化联系在一起。3.从REV反应元件确定大RNA片段的全局结构。确定HIV-1REV和REV-REV在RNA上的结合是否会导致螺旋的重排。这项研究的结果将揭示螺旋连接的结构如何指导遥远的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理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，并有助于针对RNA靶标的药物的开发。

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.