ANALYSIS OF RATE-DETERMINING CONFORMATIONAL-CHANGES DURING SELF-SPLICING OF THE TETRAHYMENA INTRON

RNA catalyzed reactions are often limited in vitro by the rate of stru ctural rearrangements in the RNA. Analysis of intra- and intermolecula r splicing of the Tetrahymena preribosomal RNA revealed two well resol ved kinetic phases with rate constants of approximately 2.5 and 0.02 m in(-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 mu tations result in redistribution of the RNA among different conformati ons 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 de grees C (0.3 -0.5 min(-1)). In contrast, the intermediate complex afte r 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 p rovide an initial framework for studying conformational changes that a ccompany excision of the Tetrahymena intron from ribosomal RNA.