STRUCTURAL ORGANIZATION OF RIBOSOMES AND LARGE RNAS

通过peptiens和其他试剂的交联将用于绘制 游离和核糖体结合的rRNA的二级和三级结构。 初步工作将集中在E。coli16SrRNA。 特别感兴趣的 是多核苷酸链的远距离区域之间的接触。 通过电子显微镜和凝胶电泳分析这些区域之间的交联 电泳 这些技术的改进将大大加快 分析RNA结构，并允许直接比较折叠的 细菌、细胞器和真核生物中rRNA的结构。 新的快速 技术将被开发，以允许交叉连接的位置， 定位在核酸序列的水平上。 交联还将 用于分析6种RNA之间的相互作用， 核糖体 我们还将开发技术来检查 RNA中的打结拓扑。 化学修饰、交换反应和 X射线散射将被用来检查核糖体结合的结构， mRNA。 将开发印迹技术以进行交联研究 不经纯化的RNA。 这些应具有广泛的适用性， RNA剪接、肿瘤病毒RNA结构及 在转录、加工和翻译过程中的特定mRNA。 的 所有这些项目背后的理由是最近的认识， 分离的大RNA可以形成稳定的有趣的紧凑折叠 结构图 远距离接触是唯一的信息n 建立和比较这些结构。 简单的屏幕， 接触可能会深刻影响目前的研究，在生物物理 调控化学与分子生物学、肿瘤病毒与蛋白质 合成.

Crosslinking by psoralens and other reagents will be used to map aspects of the secondary and tertiary structure of free and ribosome-bound rRNAs. Initial work will focus on the E. coli 16S rRNA. Of particular interest are contacts between distant regions of the polynucleotide chain. Crosslinks between such regions are analyzed by electron microscopy and gel electrophoresis. Improvements in these techniques should greatly speed the analysis of RNA structure and allow direct comparison of the folded structures of rRNAs in bacteria, organelles and eukaryotes. New rapid techniques will be developed to allow the positions of crosslinks to be localized a the level of the nucleic acid sequence. Crosslinking will also be used to analyze for interactions between the 6 RNAs on a functioning ribosome. We will also develop techniques for examining the presece of knotted topologies in RNAs. Chemical modification, exchange reactions and x-ray scattering will be used to examine the structure of ribosome-bound mRNA. Blotting techniques will be developed to allow crosslinking studies of RNAs without purification. These should have broad applicability to studies on RNA splicing, tumor virus RNA structure and the structure of particular mRNA during transcription, processing and translation. The rationale behind all of these projects is the recent realization that isolated large RNAs can form stable interesting compact folded stgructures. Long distance contacts are uniquely informative n establishing and comparing such structures. Simple screens for such contacts could profoundly influence current research in the biophysical chemistry and molecular biology of regulation, tumor viruses and protein synthesis.