Group i splicing in vitro and in vivo

Competition between alternative RNA structures is important for understanding the folding kinetics and function of RNA. The self-splicing group I intron front Tetrahymena rDNA is a useful model for RNA folding, because its autocatalytic activity is directly related to its three-dimensional structure. Self-splicing is attenuated in vitro by partitioning of the RNA population between active and inactive conformations at 30 C. The equilibrium betweeu active and inactive pre-rRNA structures is affected by exon sequences distant from the 5' splice site, suggesting that intron excision is also coupled to correct folding of the rRNA. Point mutations in the 5' exon can promote misfolding of a domain of the intron that is essential for catalytic activity. Both the rate and the yield of active pre-rRNA is increased by higher temperatures or by moderate concentrations of denaturant, in agreement with the idea that folding of this transcript is limited by incorrect structures in vitro. In vivo, the rate of splicing is 50-fold faster than it is in vitro, suggesting that proteins or other factors promote correct folding of the pre-rRNA. The Tetrahymena intron is rapidly spliced when inserted into the homologous position of the E. coli 23S rRNA. suggesting that in vivo splicing of this intron does not require a specific splicing factor, but rather than common cellular components are sufficient to accelerate RNA catalyzed reactions.