Fig. 2: Effect of Mg²⁺ and F^- on the folding of the riboswitch (RNA64).

FRET histograms and corresponding TODPs in the absence (a) and presence (b) of F^-. Histograms are well fitted with three Gaussian peaks, shown in green, red and cyan for the low-, mid- and high-FRET states, respectively, with the cumulative fit shown in blue. The percent population of all fitted peaks are shown in respective colors in each histogram panel, and the number of molecules that were analyzed is indicated by “N”. TODPs represent dynamic traces as ‘off-diagonal’ and static traces as ‘on-diagonal’ features, where the color scale shows the prevalence of each population. c Percent of all traces that remain statically in the mid-FRET (docked) state as a function of Mg²⁺concentration in the absence (red) and presence (blue) of 1 mM F^-. d, e Kinetics of low- to mid-FRET and high- to mid-FRET transitions as a function of Mg²⁺ concentration. P, U, D stand for prefolded (high-FRET), unfolded (low-FRET) and docked (mid-FRET) states, respectively. The black diamond and red triangle symbols represent rates in 2 mM Mg²⁺ and 1 mM F^-. The error bars are presented as ± standard deviation for n = 2 independent experimental datasets. f Representative smFRET traces during real-time ligand jump experiments at 1 mM Mg²⁺ (left two traces) and at 2 mM Mg²⁺ (right two traces) before and after the addition of 1 mM F^-. Upon addition of F^- molecules transition from the DD to the SD conformation. The time of F^- addition is indicated by the green arrow.