Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding

We distinguish the contribution of the electrostatic environment, initial conformational ensemble, and macromolecular stability on the folding mechanism of a large RNA using a combination of time-resolved "Fast Fenton" hydroxyl radical footprinting and exhaustive kinetic modeling. This integrated approach allows us to define the folding landscape of the L-21 Tetrahymena thermophila group I intron structurally and kinetically from its earliest steps with unprecedented accuracy. Distinct parallel pathways leading the RNA to its native form upon its Mg²⁺-induced folding are observed. The structures of the intermediates populating the pathways are not affected by variation of the concentration and type of background monovalent ions (electrostatic environment) but are altered by a mutation that destabilizes one domain of the ribozyme. Experiments starting from different conformational ensembles but folding under identical conditions show that whereas the electrostatic environment modulates molecular flux through different pathways, the initial conformational ensemble determines the partitioning of the flux. This study showcases a robust approach for the development of kinetic models from collections of local structural probes.