PHYSICAL AND GENETIC STUDIES OF RNA STRUCTURES

本建议的目的是详细研究结构和动力学 E. coli 16S核糖体RNA，识别S4 蛋白质（大于500个碱基）或S8和S15蛋白质（大约200个碱基 基地）。 这项工作的目的是了解原则 控制大RNA分子的折叠和相互作用。 三 将采用补充办法： RNA结构的中分辨率作图：特定RNA片段 将制备覆盖感兴趣区域的样品，并使用结构 描述RNA二级结构的特异性试剂， 主要的三级相互作用和与结合蛋白的接触点。 将片段的蛋白结合亲和力与完整的16S rRNA。 详细的物理研究：一旦一个相当小的RNA片段（小于 150个碱基）被发现具有特别有趣的结构或蛋白质 结合的特点，它将大量准备的研究，通过光学 和核磁共振光谱，以及其结晶的x射线衍射研究 将尝试。 这些物理方法能够提供一个非常 高分辨率图片的折叠，相互作用，和动态的一个 有限的RNA分子。 第一批研究将使用112个碱基的片段 含有S15结合位点，已知具有异常高的亲和力 嵌入剂的位置。 遗传学研究：将制定选择突变的一般方案 缺乏蛋白质-RNA识别;这些将用S4进行 蛋白质和核糖体和信使RNA能够特异性地结合它。 突变位点的定位将确定必需的RNA和蛋白质 具有约束力的功能，并将物理研究导向重要区域。

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.