Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.     

Counterion charge density determines the position and plasticity of RNA folding transition states

The self-assembly of RNA structure depends on the interactions of counterions with the RNA and with each other. Comparison of various polyamines showed that the tertiary structure of the Tetrahymena ribozyme is more stable when the counterions are small and highly charged. By monitoring the folding kinetics of the ribozyme as a function of polyamine concentration, we now find that the charge density of the counterions determines the positions of the folding transition states. The transition state ensemble (TSE) between U and N moves away from the native state as the counterion valence and charge density increase, as predicted by the Hammond postulate. The TSE is broader and less structured when the RNA is refolded in polyamines rather than Mg2+. That the charge density of the counterions determines the plasticity of the TSE demonstrates the importance of interactions among condensed counterions for the self-assembly of RNA structures. We propose that the major barrier to RNA folding is dominated by entropy changes when counterion charge density is low and enthalpy differences when it is high.