A summary of the analyzed folding reactions. The “unfolded” wild-type RNA pictures represent one of the ensemble of conformers consistent with the ·OH reactivity pattern and Stokes radius measured at the indicated experimental condition (SI Figs. 5 and 6). The Mg²⁺-folded ribozyme model is from ref. . Reactions A and B (solid arrows) are folding experiments in which Mg²⁺ is added over a constant concentration of Na⁺ or K⁺. In reaction C (dashed arrow), Mg²⁺ and either Na⁺ or K⁺ are added simultaneously to initiate folding. Experiments with the L5b mutant ribozyme were conducted as described above.

Summary of time–progress curves for L-21 T. thermophila group I intron and their clustering. (A) Average time–progress curves summarizing the clusters distinguished by k-means clustering of the 25 ribozyme protections analyzed for each folding reaction: reactions A and B () are 200 mM Na⁺ (solid line), 600 mM Na⁺ (open squares), 200 mM K⁺ (dashed line), and 600 mM K⁺ (filled circles); reaction C is 200 mM Na⁺ (filled circles) and K⁺ (filled squares) adjusted to 600 mM concurrently with Mg²⁺. The clusters are characterized by fast (green), medium (red), and slow (blue) initial rates. The secondary structure insets show the structural localization of the clusters. The data were smoothed by Golay interpolation for easier visualization although the raw data were analyzed. (B) Clustograms summarizing the 25 tertiary contacts analyzed for each of different folding experiments. The fast, medium, and slow clusters are colored red, green, and blue for the wild type and magenta, yellow, and cyan for the mutant ribozymes, respectively. (C) Time–progress curves for the L5b mutant ribozyme as described in A for the wild type. The clusters are characterized by fast (magenta), medium (yellow), and slow (cyan) initial rates. The alternate coloring schemes reflect the unique structural characters of the mutant and wild-type ribozyme intermediates.

The kinetic models resolved for the Mg²⁺-induced folding of wild-type (A) and L5b (B) ribozymes. The structured regions of each species are modeled from the ·OH reactivity patterns and rendered as bold ribbons. These models are representative conformations, as the unstructured regions can potentially adopt alternative conformations. The thickness of the arrows is proportional to the resolved rate. The reverse arrows for the U→Ii and Ii→F reactions are omitted for clarity. Black arrows indicate that there is no significant difference in rate measured for the different salt concentrations. Gray-black arrows indicate differences in the resolved rates between the two salt concentrations indicated (black and gray represent 200 and 600 mM Na⁺, respectively). The rates that are dramatically different for folding in the presence of K⁺ rather than Na⁺ are shown by black dashed arrows. The numbers accompanying arrows indicate the resolved rates (in s⁻¹) for the folding transition conducted in the presence of 200 mM Na⁺. The other rates, including those for K⁺-containing solutions, are summarized in SI Figs. 15 and 16.

Time evolution of the intermediates (I1, I2, and I3) and the final state (F) observed for folding reactions A (cyan), B (magenta), and C (black) outlined in . The width of the curves illustrates propagated errors on the time evolution of the intermediates. (Insets) The relative initial fluxes and propagated error for each reaction calculated from the ratios of the measured rate constants.