ORGANIZATION AND FUNCTION OF THE E COLI 16S RIBOSOMAL RN

长期目标是确定的三维结构的 e. coli16SrRNA在工作核糖体中的表达。 这一目标包括确定 RNA结构的变化是否发生在蛋白质合成过程中， 这样的话，它包括了对不同状态的描述。 这 这种方法应该确定核糖体中的基因座， 功能 在本供资期间， 核糖体中16 S rRNA内的长距离接触将是 研究并鉴定活性位点的RNA。 这些 研究将以适当的方式完善几个领域的结构， 16S rRNA的三维模型，并将有助于确定是否 这些位点的相互作用在功能上是重要的。 这项工作是 因为E。大肠杆菌核糖体已被广泛研究， 对蛋白质翻译的理解将取决于详细的 组件的结构分析以及它们如何物理相互作用。 在 与此同时，描述翻译的模型变得越来越清晰， 真细菌中的翻译调节也将适用于 真核生物 将采取这些方法：（1）确定是否有 16S rRNA中长距离相互作用的差异， 与亚基的功能状态相关。 光化学磷 交联和变性凝胶电泳分析将用于 检测RNA内接触。 可能有必要直接将pecuen 单加合物的特定位点，以获得灵敏度和避免 杀死RNA的生物活性。 (2)确定位置， 30S核糖体中活性位点的边界，通过使用合成或 携带光化学试剂的天然mRNA。 (3)修饰核糖体RNA 和结构/功能相关性测试： 由A博士制造的改变的16S rRNA的结构。Dahlberg及其同事（Brown 大学）;通过将DNA寡核苷酸连接在 选择性地影响其功能活性的几个选定位点;使 16SrRNA体外转录，并将其重组为活性 亚单位 如果最后一次实验成功，那么选择性改变 基因内的区域通过体外操作。

The long range goal is to determine the three-dimensional structure of the E. coli 16S rRNA in the operating ribosome. This goal includes determining whether changes in the RNA structure occur during protein synthesis and, if this is so, it includes a description of the different states. This approach should identify the loci in the ribosome that define its function. In the present funding period the occurrence of several long-distance contacts within the 16S rRNA in the ribosome will be investigated and the RNA at the active site will be identified. These studies will refine the structure of several areas in an appropriate three-dimensional model of the 16S rRNA and will help determine whether the interactions at these sites are functionally important. This work is important because the E. coli ribosome has been extensively studied and an understanding of protein translation will depend upon the detailed structural analysis of the components and how they physically interact. At the same time, it is becoming clearer that models that describe translation and the regulation of translation in eubacteria will also be applicable to eukaryotes. These approaches will be taken: (1) Determine whether there are differences in the long distance interactions in the 16S rRNA which are correlated to the functional state of the subunit. Psoralen photochemical crosslinking and analysis by denaturing gel electrophoresis will be used to detect intra RNA contacts. It may be necessary to direct psoralen monoadducts to specific sites in order to gain sensitivity and to avoid killing the biological activity of the RNA. (2) Identify the position and boundaries of the active sites in the 30S ribosome, by using synthetic or natural mRNA carrying photochemical reagents. (3) Modify the ribosomal RNA and test for structural/functional correlations: characterize the structure of altered 16S rRNA made by Dr. A. Dahlberg and co-workers (Brown University); alter the 16S rRNA by attaching DNA oligonucleotides at several chosen sites to selectively affect its functional activity; make 16S rRNA by in vitro transcription and reconstitute it into an active subunit. If the last experiment is successful, then selectivity alter regions within the gene by in vitro manipulations.