FOLDING STUDIES OF A GROUP I INTRON CATALYTIC RNA

在过去的十年里，人们认识到RNA能够催化 强调了RNA在许多细胞过程中的关键作用。 嗜热四膜虫Ⅰ组内含子RNA是第一个 被发现具有催化能力， 广泛研究的RNA。在体内，它进行自我剪接反应。 在体外，它折叠成独特的三维结构， 在催化过程中构象发生变化，并能够底物 识别和结合，以及辅因子结合。尽管数量巨大 关于RNA的三级结构和催化作用的研究， RNA折叠的实际机制。该提案的目的是利用 时间分辨X射线羟自由基足迹技术 研究四膜虫内含子的折叠。该技术 能够在几十毫秒的时间尺度上分辨折叠事件， 单碱基分辨率。该提案可分为两大类 目标.第一个目标是描述孤立的 RNA的“激活剂”结构域--P5 abc区域--及其随后的对接 剩下的RNA。第二个目标是调查 动力学陷阱对褶皱动力学和机制的影响 在几种快速折叠突变体的折叠之后， 变性剂来加速折叠过程。

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.