PHYSICAL AND GENETIC STUDIES OF RNA STRUCTURES

这项建议的目的是详细研究结构和动力学 在大肠杆菌16S核糖体RNA中的两个区域，识别S4 蛋白质(大于500个碱基)或S8和S15蛋白质(约200个碱基 基地)。这项工作的目标是理解原则 管理大的RNA分子的折叠和相互作用。三 将使用互补的方法： RNA结构的中等分辨率作图：特定的RNA片段 将准备覆盖感兴趣的区域，并用结构进行探索 用于描绘RNA二级结构、区域的特定试剂 主要的三级相互作用，以及与结合蛋白的接触点。 片段的蛋白质结合亲和力将与完整的16S进行比较 RRNA。 详细的物理研究：一旦相当小的RNA片段(少于 150个碱基)具有特别有趣的结构或蛋白质 结合特性，将大量制备用于光学研究 和核磁共振谱及其结晶用于X-射线衍射研究 将会被尝试。这些物理方法能够给出一个非常 高分辨率的折叠、相互作用和动态图像 有限的RNA分子。最初的研究将是112个碱基片段 含有S15结合位点，并且已知具有异常高的亲和力 嵌入器的场地。 基因研究：将制定选择突变的一般方案 缺乏蛋白质-RNA识别；这些将通过S4进行 蛋白质、核糖体和信使RNA能够特异性地结合它。 突变部位的图谱将确定必要的RNA和蛋白质 用于结合的特征，并将物理研究引导到重要区域。

The aim of this proposal is to study in detail the structure and dynamics of two regions in the E. coli 16S ribosomal RNA, recognizing either S4 protein (greater than 500 bases) or S8 and S15 proteins (approximately 200 bases). The objective of the work is to understand the principles governing the folding and interaction of large RNA molecules. Three complementary approaches will be used: Medium resolution mapping of RNA structures: specific RNA fragments covering the regions of interest will be prepared and probed with structure specific reagents to delineate the RNA secondary structures, regions of major tertiary interactions, and points of contact with bound proteins. The protein binding affinity of fragments will be compared to intact 16S rRNA. Detailed physical studies: once a fairly small fragment of RNA (less than 150 bases) is found to have particularly interesting structural or protein binding features, it will be prepared in quantity for studies by optical and NMR spectroscopy, and its crystallization for x-ray diffraction studies will be attempted. These physical methods are capable of giving a very high resolution picture of the folding, interactions, and dynamics of a limited RNA molecule. The first studies will be with a 112 base fragment containing the S15 binding site and known to have unusually high affinity sites for intercalators. Genetic studies: General schemes will be devised for selecting mutations deficient in a protein - RNA recognition; these will be carried out with S4 protein and the ribosomal and messenger RNAs able to bind it specifically. Mapping of the mutation sites will define the essential RNA and protein features for binding and direct the physical studies to important regions.