STRUCTURAL ORGANIZATION OF RIBOSOMES AND LARGE RNAS

补骨脂素和其他试剂的交联剂将被用来定位 游离和核糖体结合的rRNA的二级和三级结构。 最初的工作将集中在大肠杆菌16S rRNA上。特别感兴趣的 是多核苷酸链远距离区域之间的接触。 用电子显微镜和凝胶分析了这些区域之间的交联物。 电泳法。这些技术的改进应该会大大加快 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。新快速 将开发技术以允许将交联物的位置 定位了核酸序列的水平。交联剂也将 用于分析运行中的6个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。化学改性、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剂研究 在没有纯化的情况下。这些应该具有广泛的适用性 RNA剪接、肿瘤病毒RNA结构及病毒结构的研究 转录、加工和翻译过程中的特定mRNA。这个 所有这些项目背后的理由是最近意识到 分离的大RNA可以形成稳定的、有趣的紧凑折叠 结构。长途联系人具有独一无二的信息量 建立和比较这样的结构。简单的屏幕，这样的 接触可以深刻地影响当前生物物理领域的研究 调控、肿瘤病毒和蛋白质的化学和分子生物学 综合。

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.