Time Recovered Hydroxyl Radical Footprinting of RNA

描述（由申请人提供）：在所有生物体中，RNA分子都需要将基因翻译成蛋白质，并打开和关闭特定基因。控制RNA的功能障碍与肌肉萎缩症、脆性X染色体智力低下和某些肿瘤等疾病有关。为了正常发挥功能，RNA必须折叠成特定的三维形状，并且必须与特定的蛋白质组装。例如，核糖体包含两个大的RNA和50多个蛋白质，它们必须以正确的方式聚集在一起。这项研究的目标是了解RNA如何折叠，以及它们如何与蛋白质合作形成细胞“机器”，如核糖体。这些结果将有助于了解某些RNA在人类遗传疾病中的功能，并将有助于设计抗菌和抗病毒药物。我们以前使用的时间分辨羟基自由基足迹，小角散射和生化方法来探测核酶的折叠途径。最近，我们已经使用足迹跟踪SOS核糖体的组装在真实的时间。在第一个目标中，一个稳定的细菌组I核酶将被用作一个模型系统，以解剖相互作用，合作稳定RNA的三级结构。目的2-4将从核酶研究中获得的RNA折叠原理应用于SOS核糖体的组装。通过保护RNA骨架免受羟基自由基裂解和通过标记RNA的荧光变化来监测RNA和RNA-蛋白质相互作用的形成。要解决的问题是（1）蛋白质如何识别其结合位点并稳定rRNA中的三级相互作用，（2）蛋白质是否改变核糖体RNA折叠途径，以及（3）前168 rRNA的折叠和组装。长期目标是建立核糖体原位组装的测定方法，并了解RNA折叠的保真度，前rRNA的组装和加工之间的联系。

DESCRIPTION (provided by applicant): In all organisms, RNA molecules are needed to translate genes into protein, and to turn specific genes on and off. The malfunction of controlling RNAs has been linked to diseases such as muscular dystrophy, Fragile X mental retardation, and certain tumors. In order to function normally, RNAs must fold into specific three-dimensional shapes and must assemble with particular proteins. For example, the ribosome contains two large RNAs and more than 50 proteins which must come together in precisely the right way. The goal of this research is to understand how RNAs fold up, and how they cooperate with proteins to form cellular "machines" such as the ribosome. The results will help understand how certain RNAs malfunction in human, genetic diseases, and will assist the design of antibacterial and antiviral drugs. We have previously used time-resolved hydroxyl radical footprinting, small angle scattering and biochemical methods to probe the folding pathway of ribozymes. Recently, we have used footprinting to follow the assembly of the SOS ribosome in real time. In the first aim, a stable bacterial group I ribozyme will be used as a model system to dissect the interactions that cooperatively stabilize RNA tertiary structure. The principles of RNA folding obtained from studies of ribozymes will be applied to assembly of SOS ribosomes in aims 2-4. The formation of RNA and RNA-protein interactions will be monitored by protection of the RNA backbone from hydroxyl radical cleavage and by changes in the fluorescence of labeled RNAs. Questions to be addressed are (1) how proteins recognize their binding sites and stabilize tertiary interactions in the rRNA, (2) whether proteins change the ribosomal RNA folding pathway, and (3) folding and assembly of the pre-168 rRNA. Long-term goals are to establish assays for ribosome assembly in situ, and to understand the link between the fidelity of RNA folding, assembly and processing of the pre-rRNA.