Folding intermediates of a self-splicing RNA: Mispairing of the catalytic core

The Tetrahymena thermophila self-splicing RNA is trapped in an inactive conformation during folding reactions at physiological temperatures. The structure of this metastable intermediate was probed by chemical modification interference and site-directed mutagenesis. In the inactive structure, an incorrect base-pairing, which we call Alt P3, displaces the P3 helix in the catalytic core of the intron. Mutations that stabilize Alt P3 increase the fraction of pre-rRNA that becomes trapped in the inactive structure, whereas mutations that destabilize Alt P3 reduce accumulation of this conformer. At high concentrations of Mg²⁺, the yield of correctly folded mutant pre-rRNAs is similar to wild-type RNA. Under these conditions, the rate of folding for mutant RNAs is slower than for the wild-type, but is increased by addition of urea. The results show that slow folding of the Tetrahymena pre-rRNA is a consequence of non-native secondary structure in the catalytic core of the intron, which is linked to an alternative hairpin in the 5' exon. This illustrates how kinetically stable, long-range interactions shape RNA folding pathways.