Time Recovered Hydroxyl Radical Footprinting of RNA

描述(申请人提供)：在所有生物体中，RNA分子都需要将基因转化为蛋白质，并打开和关闭特定的基因。控制RNA的功能障碍与肌肉营养不良、脆性X智力低下和某些肿瘤等疾病有关。为了正常运作，RNA必须折叠成特定的三维形状，并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多种蛋白质，它们必须以正确的方式结合在一起。这项研究的目标是了解RNA是如何折叠的，以及它们如何与蛋白质合作形成核糖体等细胞“机器”。这一结果将有助于理解某些RNA如何在人类遗传性疾病中发挥作用，并将有助于抗菌和抗病毒药物的设计。我们已经使用时间分辨的羟基自由基足迹、小角散射和生化方法来探索核酶的折叠途径。最近，我们利用足迹技术实时跟踪SOS核糖体的组装。在第一个目标中，一个稳定的细菌I组核酶将被用作一个模型系统来剖析协同稳定RNA三级结构的相互作用。从核酶研究中获得的RNA折叠原理将应用于AIMS 2-4中SOS核糖体的组装。RNA和RNA-蛋白质相互作用的形成将通过保护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。需要解决的问题是(1)蛋白质如何识别它们的结合部位并稳定rRNA中的三级相互作用，(2)蛋白质是否改变了核糖体RNA的折叠途径，以及(3)Pre-168 rRNA的折叠和组装。长期目标是建立核糖体原位组装的分析方法，并了解RNA折叠、组装和前rRNA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.