ORGANIZATION AND FUNCTION OF THE E COLI 16S RIBOSOMAL RN

长期的目标是确定 E.Coli16S核糖体中的rRNA。这一目标包括确定 在蛋白质合成过程中是否发生了RNA结构的变化，如果 是这样的，它包括对不同状态的描述。这 这种方法应该确定核糖体中定义其 功能。在本供资期间，发生了几起 核糖体中16S rRNA内的远程接触将是 调查后，活性部位的RNA将被鉴定。这些 研究将在适当的情况下完善几个领域的结构 16S rRNA的三维模型，将有助于确定 这些网站的互动在功能上很重要。这项工作是 重要的是因为大肠杆菌核糖体已经被广泛研究，并且 对蛋白质翻译的理解将取决于详细的 对组件及其物理交互方式进行结构分析。在… 与此同时，描述翻译的模型正变得越来越清楚 而在真细菌中的翻译规则也将适用于 真核生物。 将采取以下方法：(1)确定是否有 16S rRNA的长距离相互作用的差异是 与亚基的功能状态相关。补骨脂素光化学 将采用交联法和变性凝胶电泳法分析 检测RNA内接触情况。可能有必要指导补骨脂素 单加合物到特定的位置，以获得敏感性和避免 杀死核糖核酸的生物活性。(二)找准岗位， 30S核糖体中活性部位的边界，通过使用合成或 天然信使核糖核酸携带光化学试剂。(3)对核糖体RNA进行修饰 和结构/功能相关性的测试：描述 A.Dahlberg博士及其同事(Brown)制作的改变的16S rRNA的结构 大学)；通过将DNA寡核苷酸连接到 有选择地影响其功能活动的几个地点；使 16S rRNA体外转录并将其重组为具有活性的 亚单位。如果上一次实验成功，那么选择性改变 通过体外操作获得基因内的区域。

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.