Global Structures of RNA

描述（由申请人提供）：为了实现其多样化的生物学功能，RNA分子必须折叠成特定的三级结构，以产生催化中心或配体识别位点。尽管最近出现了大量关于RNA分子和RNA-蛋白复合物的三维结构的信息，最终以核糖体颗粒的结构为高潮，但RNA采用这些结构的机制尚不清楚。这项研究的长远目标是了解RNA折叠的机制。RNA折叠的基本特征包括构象搜索、金属离子结合和生成中间体。此外，大多数大RNA分子需要蛋白质辅助因子才能正确有效地折叠。因此，我们将继续分析发夹核酶的三级结构形成，这是一种自主折叠的RNA，我们也将对哺乳动物信号识别颗粒（SRP）的7SL RNA的折叠进行新的研究，SRP在多种蛋白质伴侣存在下折叠。通过研究这两个系统，我们希望了解RNA折叠的一般原理。此外，该结果将有助于设计用于基因治疗的改良核酶，并最终可能导致基于SRP机制阻止蛋白质合成的新的癌症治疗方法。具体目的是：(1)阐明环重排和动态环结构在发夹核酶三级结构形成中的作用。(2)测试提出的发夹核酶三级结构形成的构象搜索模型，并确定搜索的决定因素。(3)监测信号识别颗粒的Alu结构域组装过程中的RNA折叠转变。(4)阐明信号识别颗粒s结构域的初始组装机制，并表征相关的RNA折叠转变。为了实现这些目标，我们将应用一系列集合和单分子荧光方法来解剖RNA折叠途径，并监测折叠过程中的动态构象转变。此外，本项目开发的实验方法将为研究RNA的构象动力学和折叠提供新的工具，具有广泛的适用性。

DESCRIPTION (provided by applicant): To achieve their diverse range of biological functions, RNA molecules must fold into specific tertiary structures that create catalytic centers or ligand recognition sites. Despite the recent emergence of a wealth of information on the three-dimensional structures of RNA molecules and RNA-protein complexes, culminating in structures of ribosomal particles, the mechanisms by which RNA adopts these structures are poorly understood. The broad, long-term objective of this proposal is to understand the mechanism of RNA folding. Fundamental features of RNA folding include conformational search, metal ion binding and productive intermediates. Additionally, most large RNA molecules require protein cofactors in order to fold correctly and efficiently. Hence, we will continue to analyze tertiary structure formation in the hairpin ribozyme, an autonomously folding RNA, and we will also undertake new studies of the folding of the 7SL RNA from the mammalian signal recognition particle (SRP), which folds in the presence of multiple protein chaperones. By studying both systems, we hope to learn about the general principles of RNA folding. Moreover, the results will facilitate the design of improved ribozymes for therapeutic applications in gene therapy and, eventually, may lead to new cancer therapies based on arrest of protein synthesis by the SRP machinery. The specific aims are: (1) Elucidate the role of loop rearrangements and dynamic loop structure in tertiary structure formation in the hairpin ribozyme. (2) Test the proposed conformational search model for tertiary structure formation in the hairpin ribozyme and identify determinants of the search. (3) Monitor RNA folding transitions during assembly of the Alu domain of the signal recognition particle. (4) Elucidate the mechanism of the initial assembly of the S-domain of the signal recognition particle and characterize associated RNA folding transitions. To address these goals, we will apply a range of ensemble and single-molecule fluorescence methods to dissect the RNA folding pathways and to monitor dynamic conformational transitions during folding. Additionally, the experimental methods developed during this project will provide new tools for studying the conformational dynamics and folding of RNA and will be broadly applicable.