Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

RNA is commonly believed to undergo a number of sequential folding steps before reaching its functional fold, i.e., the global minimum in the free energy landscape. However, there is accumulating evidence that several functional conformations are often in coexistence, corresponding to multiple (local) minima in the folding landscape. Here we use the 5′-exon–intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg2+ and Ca2+ on RNA tertiary structure formation. Bulk and single-molecule spectroscopy reveal that near-physiological M2+ concentrations strongly promote interstrand association. Moreover, the presence of M2+ leads to pronounced kinetic heterogeneity, suggesting the coexistence of multiple docked and undocked RNA conformations. Heterogeneity is found to decrease at saturating M2+ concentrations. Using NMR, we locate specific Mg2+ binding pockets and quantify their affinity toward Mg2+. Mg2+ pulse experiments show that M2+ exchange occurs on the timescale of seconds. This unprecedented combination of NMR and single-molecule Förster resonance energy transfer demonstrates for the first time to our knowledge that a rugged free energy landscape coincides with incomplete occupation of specific M2+ binding sites at near-physiological M2+ concentrations. Unconventional kinetics in nucleic acid folding frequently encountered in single-molecule experiments are therefore likely to originate from a spectrum of conformations that differ in the occupation of M2+ binding sites.