FOLDING STUDIES OF A GROUP I INTRON CATALYTIC RNA

近十年来人们对RNA具有催化作用的认识 强调了RNA在许多细胞过程中扮演的关键角色。 嗜热四膜虫第一组内含子RNA是第一个RNA 被发现具有催化能力，并已成为世界上最 对RNA进行了广泛的研究。在体内，它会进行自我剪接反应。 在体外，它折叠成一个独特的三维结构，经历 在催化过程中的构象变化，并能够底物 识别和结合，以及辅因子结合。尽管数量巨大 在对RNA三级结构和催化作用的研究中，人们对此知之甚少 RNA折叠的实际机制。这项提案的目标是使用 时间分辨x射线羟基自由基足迹技术 研究四膜虫内含子的折叠。这项技术是 能够在几十毫秒的时间尺度上分辨折叠事件，并 具有单基分辨率。这项建议可分为两大部分 目标。第一个目标是描述孤立的 RNA的“激活”结构域--P5abc区--及其随后的对接 进入核糖核酸的其余部分。第二个目标是调查 动力学陷阱对褶皱动力学及机制的影响 在几个快速折叠突变体的折叠之后，通过使用 变性剂可以加速折叠过程。

The realization during the past decade that RNA is capable of catalysis has underscored the key role that RNA plays in many cellular processes. The Tetrahymena thermophila group I intron RNA was the first RNA discovered to have catalytic ability and has been one of the most extensively studied RNAs. In vivo it carries out a self-splicing reaction. In vitro, it folds into a distinct three dimensional structure, undergoes conformational changes during catalysis, and is capable of substrate recognition and binding, and of co-factor binding. Despite the huge number of studies on RNA tertiary structure and catalysis, little is known about the actual mechanisms of RNA folding. The goal of this proposal is to use the technique of time-resolved x-ray hydroxyl radical footprinting to investigate the folding of the Tetrahymena intron. This technique is capable of resolving folding events on tens of millisecond timescales and with single base resolution. The proposal can be divided into two broad goals. The first goal is to characterize the folding of the isolated "activator" domain of the RNA--the P5abc region--and it subsequent docking into the remainder of the RNA. The second goal is to investigate the effect of kinetic traps on the folding dynamics and mechanisms by following the folding of several fast-folding mutants, and by using denaturants to accelerate the folding process.