Time Resolved Hydroxyl Radical Footprinting of RNA

描述（由申请人提供）：RNA分子和RNA-蛋白质复合物是动态结构，这种运动是其功能所固有的。对RNA折叠机制的研究将为基因调控过程中RNA蛋白复合物的组装以及催化过程中发生的构象变化提供物理见解。虽然RNA的二级和三级相互作用很快形成，但有些RNA很容易陷入错误折叠状态。如果RNA发生故障，替代构象之间的竞争可能导致疾病，或者可以用于调节基因活性和RNA病毒的复制。因此，从其一级序列理解和预测RNA功能的能力对于分析和治疗遗传疾病以及开发靶向RNA分子的治疗剂是重要的。一个小的组I核酶从Azoarcus的折叠机制将比较折叠的较大的四膜虫核酶。这是研究RNA结构组装早期步骤的理想系统，因为超过一半的RNA在100 ms内折叠，并且三级结构被许多类型的金属离子稳定。使用同步加速器X射线束的羟基自由基足迹法将用于以20 ms的间隔检测RNA三级结构的变化。这种方法是独特的，因为它解决了RNA中特定位点的构象变化。停流荧光光谱法将用于监测含有2-氨基嘌呤和荧光素的RNA的全局折叠，分辨率为1 ms。拟议的研究解决序列和金属离子的相互作用如何直接组装特定的三级结构天然构象。时间分辨羟基自由基足迹法也将用于研究RNA-蛋白质复合物的组装，使用细菌RNase P全酶作为测试系统。RNase P是所有细胞中存在的必需酶。RNA亚基的折叠途径与I组核酶的折叠途径有许多相同的性质，但尚不清楚蛋白质亚基如何改变催化活性RNA的形成。由于X射线很容易穿透细胞和整个组织，这种方法将适用于研究体内I组核糖核酸酶和RNase P的三维结构。

DESCRIPTION (provided by applicant): RNA molecules and RNA-protein complexes are dynamic structures, and this motion is intrinsic to their function. The study of RNA folding mechanisms will provide physical insight into the assembly of RNAprotein complexes during gene regulation, and conformational changes that take place during catalysis. Although RNA secondary and tertiary interactions form quickly, some RNAs are easily trapped in misfolded states. Competition among alternative conformations can lead to disease if RNAs malfunction, or can be exploited for regulation of gene activity and replication of RNA viruses. Thus, the ability to understand and predict RNA function from its primary sequence is important to the analysis and treatment of genetic disease, and the development of therapeutic agents that target RNA molecules. The folding mechanism of a small group I ribozyme from Azoarcus will be compared with folding of the larger Tetrahymena ribozyme. This is an ideal system to study early steps in the assembly of RNA structure, because more than half the RNA folds within 100 ms, and the tertiary structure is stablilized by many types of metal ions. Hydroxyl radical footprinting using a synchrotron X-ray beam will be used to detect changes in RNA tertiary structure at 20 ms intervals. This method is unique, in that it resolves conformational changes at specific sites in the RNA. Stopped-flow fluorescence spectroscopy will be used to monitor global folding of RNAs containing 2-aminopurine and fluorescein, with 1 ms resolution. The proposed studies address how sequence and metal ion interactions direct the assembly of specific tertiary structures native conformations. Time resolved hydroxyl radical footprinting will also be used to study the assembly of RNA-protein complexes, using the bacterial RNase P holoenzyme as a test system. RNase P is an essential enzyme present in all cells. The folding pathway of the RNA subunit shares many properties with those of group I ribozymes, but it is not known how the protein subunit alters the formation of catalytically active RNA. As Xrays easily penetrate cells and whole tissues, this method will be adapted to study the three-dimensional structure of group I riboyzmes and RNase P in vivo.