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 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.
Emerick
V L
VL
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742-2021, USA.
Pan
J
J
Woodson
S A
SA
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D005091:Exons; D006160:Guanosine Triphosphate / Q000378:metabolism; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D016053:RNA, Protozoan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012322:RNA Precursors; D016053:RNA, Protozoan; D006160:Guanosine Triphosphate