Analysis of Rate-Determining Conformational Changes during Self-Splicing of the <i>Tetrahymena</i> Intron

Abstract

RNA catalyzed reactions are often limited in vitro by the rate of structural rearrangements in the RNA. Analysis of intra- and intermolecular splicing of the Tetrahymena preribosomal RNA revealed two well resolved kinetic phases with rate constants of approximately 2.5 and 0.02 min-1 at 30 degrees C. The data are consistent with a model in which the second phase results from slow refolding of the pre-rRNA. Point mutations result in redistribution of the RNA among different conformations that can be detected by native gel electrophoresis. The active pre-rRNA rapidly progresses to a product complex in the presence of GTP. Release of the ligated exons is slightly slower than splicing at 30 degrees C (0.3 -0.5 min-1). In contrast, the intermediate complex after the first step of splicing dissociates much more slowly (5 x 10(-3) min-1), accounting for the low amount of intron-3' exon intermediate typically seen during splicing of wild type pre-rRNA. These results provide an initial framework for studying conformational changes that accompany excision of the Tetrahymena intron from ribosomal RNA.