Folding of the Tetrahymena Group I Ribozyme

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。该计划项目建议通过综合应用各种方法来研究RNA折叠的机制，这些方法各自报告折叠反应的独特和互补方面，包括单分子荧光和力测量，时间分辨小角度X射线散射，时间分辨X射线“足迹”，计算和酶学。这个阿森纳的方法将集中在一个单一的RNA的折叠，一个核酶来自自我剪接组I内含子的嗜热四膜虫。四膜虫核酶在其结构、折叠和催化机制方面是最好理解的核酶，并且其大尺寸（约400个核苷酸）为研究RNA三级结构形成的基本特征提供了充足的三维结构复杂性。所提出的研究在报告全局和局部结构、单分子和整体平均值方面具有广泛的协同作用，并将进一步开发四膜虫核酶作为未来研究的范例，比较和对比其他RNA分子的行为。从该计划中获得的信息也将允许与蛋白质的折叠行为进行比较，揭示哪些特性是每类大分子特有的，哪些是两者共有的;共同的特性可能是采用特定生物活性结构的大分子的基础。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治疗干预而改变、控制或增强提供了起点。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. This Program Project proposes to investigate the mechanism of RNA folding through the integrated application of approaches that each report unique and complementary aspects of the folding reaction, including single molecule fluorescence and force measurements, time-resolved small angle x-ray scattering, time-resolved x-ray "footprinting", computation and enzymology. This arsenal of approaches will be focused on the folding of a single RNA, a ribozyme derived from the self-splicing group I intron of Tetrahymena thermophila. The Tetrahymena ribozyme is the best-understood ribozyme in terms of its structure, folding and catalytic mechanism, and its large size (approximately 400 nucleotides) provides ample three-dimensional structural complexity for investigation of fundamental features of RNA tertiary structure formation. The proposed studies have extensive synergy in their reporting of global and local structure, single molecule and ensemble averages, and will further develop the Tetrahymena ribozyme as a paradigm for future studies comparing and contrasting the behavior of other RNA molecules. The information obtained from this Program will also allow comparison with the folding behavior of proteins, revealing which properties are specific to each class of macromolecule and which are common to both; the common properties may be fundamental to macromolecules that adopt specific biologically active structures. 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.