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.
