Vl. Emerick et al., ANALYSIS OF RATE-DETERMINING CONFORMATIONAL-CHANGES DURING SELF-SPLICING OF THE TETRAHYMENA INTRON, Biochemistry, 35(41), 1996, pp. 13469-13477
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.