Project 2 Enzymatic activity and structure probing

RNA分子，像蛋白质一样，折叠成其生物功能所需的特定三维结构。然而，我们对RNA分子如何获得其天然构象的理解仍处于起步阶段。先前的研究的结构，机制，和折叠的四膜虫组I核酶使其成为一个理想的RNA进一步解剖的折叠行为和负责这种行为的分子和拓扑性质。这个组件项目调用了几种方法，一些是成熟的，另一些是对成熟技术的直接修改，还有一些是新颖的方法，来解决 观察到的这种RNA的快速静电崩溃的性质，在建立和新的折叠途径折叠中间体的结构和性质，以及管理物种之间的相互转化率的分子特征。技术包括用含有位点特异性掺入的2-氨基嘌呤的核酶的时间分辨荧光强度测量、RNA结构的时间分辨化学探测、诱变、通过螺旋延伸引入拓扑约束、表面拴系和位点特异性标记。 交联，以及使用结合和活性测量来评估 中间体、折叠过程中的动力学分配事件和折叠结果。这些和其他组件项目的数据，当被计算核心整合时，将大大促进四膜虫核酶作为理解RNA折叠和动力学行为的范例。这种理解将为与其他RNA和蛋白质的比较研究提供基础。RNA复合物，以及有机会了解更多关于RNA和蛋白质大分子基本行为的差异和相似之处。对RNA基本行为的理解为确定其行为如何通过细胞相互作用和在治疗中被改变、控制或增强提供了一个起点。 干预这些基本性质的知识将为研究RNA的细胞行为、RNA在疾病中的作用以及RNA作为药物靶点或治疗剂的潜在用途提供基础。

RNA molecules, like proteins, fold into specific three-dimensional structures that are required for their biological function. However, our understanding of how RNA molecules attain their native conformations is in its infancy. Prior studies on the structure, mechanism, and folding of the Tetrahymena group I ribozyme render it an ideal RNA for further dissection of the folding behavior and of the molecular and topological properties responsible for this behavior. This Component Project invokes several approaches, some well-established, others straightforward modifications of well-established techniques, and some novel approaches, to address the nature of the observed rapid electrostatic collapse of this RNA, the structures and properties of folding intermediates on established and new folding pathways, and the molecular features that govern the rates of interconversion between species. Techniques include time-resolved fluorescence intensity measurements with ribozymes containing 2-aminopurine site-specifically incorporated, time-resolved chemical probing of RNA structure, mutagenesis, introduction of topological constraints by helix extensions, surface tethering, and site-specific cross-linking, and the use of binding and activity measurements to assess the properties of intermediates, kinetic partitioning events during the folding process, and the folding outcome. These and the data from the other Component Projects, when integrated by the Computational Core, will greatly further the Tetrahymena ribozyme as a paradigm for understanding the folding and dynamic behavior of RNA. This understanding will provide a basis for comparative studies with other RNAs and protein.RNA complexes, as well as an opportunity to learn more about the differences and similarities in the basic behavior of RNA and protein macromolecules. An understanding of the fundamental behavior of RNA provides a starting point for determining how its behavior may be altered, controlled, or augmented by cellular interactions and in therapeutic intervention. Knowledge of these fundamental properties will provide a foundation for studies focusing on the cellular behavior of RNA, on the role of RNA in disease, and on the potential use of RNA as a drug target or therapeutic.