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

ABSTRACT: 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 Mg(2+) and Ca(2+) on RNA tertiary structure formation. Bulk and single-molecule spectroscopy reveal that near-physiological M(2+) concentrations strongly promote interstrand association. Moreover, the presence of M(2+) leads to pronounced kinetic heterogeneity, suggesting the coexistence of multiple docked and undocked RNA conformations. Heterogeneity is found to decrease at saturating M(2+) concentrations. Using NMR, we locate specific Mg(2+) binding pockets and quantify their affinity toward Mg(2+). Mg(2+) pulse experiments show that M(2+) 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 M(2+) binding sites at near-physiological M(2+) 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 M(2+) binding sites.

SUBMITTER: Kowerko D 

PROVIDER: S-EPMC4371910 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature