Kinetic/Thermodynamic/Structural Studies of RNA Folding

RNA的结构、功能和动力学之间的关联需要对结构基础有基本的了解 在RNA折叠中。这个建议的目的是阐明RNA折叠的结构基础以及如何 核酶在转录过程中折叠，这是细胞中折叠行为的模拟。细菌的RNA成分 将使用核糖核酸酶P。在过去的三年中，已经确定了这种核酶的四种晶体结构 多年来，使我们能够在理解这种大型核酶的折叠方面取得根本性的进展。要求1 将揭示沿着折叠途径的大RNA的结构确定的主要事件， 使用实验数据/分子的组合在残基水平分辨率下折叠中间体 分子和低温电子显微镜。这种“首次”的RNA折叠途径将提供 对结构形成的顺序、核心与外围之间的相互作用， 结构，以及金属离子-RNA相互作用的作用，指导折叠和调节稳定性。目标2将 比较具有相同核心的两组RNA中关键折叠中间体的结构， 不同的外围结构。目标3将确定在转录过程中改变折叠的机制， 特别是，一个突出的暂停网站介绍了一个特定的RNA聚合酶如何影响折叠和 RNA-蛋白质复合物的组装。这项广泛的研究计划将揭示RNA的结构基础 折叠，以及RNA折叠和RNA聚合酶之间的进化和机制联系。

Correlating structure, function and dynamics of RNA requires fundamental understanding of structural basis in RNA folding. The objectives of this proposal are to elucidate the structural basis of RNA folding and how ribozymes fold during transcription, a mimic of folding behavior in the cell. The RNA component of bacterial ribonuclease P will be used. Four crystal structures of this ribozyme have been determined in the last three years, enabling us to make fundamental advances in understanding the folding of this large ribozyme. Aim 1 will reveal the major events along the folding pathway of a large RNA by the structural determination of folding intermediates at the residue-level resolution using a combination of experimental data/molecular molecular and cryo-Electron Microscopy. This "first of its kind" look at RNA folding pathways will offer unprecedented insights on the order of structure formation, the interplay between core and peripheral structures, and the role of metal ion-RNA interactions that guide folding and modulate stability. Aim 2 will compare structures of key folding intermediates among two sets of RNAs that have the same core, but distinct peripheral structures. Aim 3 will identify mechanisms for altering folding during transcription, in particular, how a prominent pause site introduced by a specific RNA polymerase affects folding and the assembly of a RNA-protein complex. This broad research program will reveal the structural basis of RNA folding, and the evolutionary and mechanistic link between RNA folding and RNA polymerase.