Magnesium dependence of the SAM-II riboswitch free energy landscape demonstrates Mg²⁺ pre-organization of bound-like state.

(a) The free energy landscape as a function of the fraction of native contacts (Q) of SAM-II near physiological Mg²⁺ concentration ([Mg²⁺] = 2.0 mM). The system explores three distinct barrier-separated minima on the free energy landscape. (b) The order of secondary structure formation as a function of the fraction of all native contacts (Q) as measured by the fraction of non-local regional contacts (Q_Regional). The transition displays high cooperativity. (c) Representative structures. The representative structure corresponding to each region of the energy landscape is designated as follows: (i) ligand-bound closed (C), (ii) ligand-free partially closed (PC) including triplex interactions, (iii) ligand-free partially open (PO), (iv) ligand-free open (O), and (v) the unfolded (U) state. Green/mauve, bases in native conformations. The ligand-bound closed minimum has been characterized from the free energy profile of the folding transition of SAM-bound RNA which indicates the ligand-free partially closed conformation has a substantial resemblance with the ligand-bound closed state. (d) Mg²⁺ concentration dependence of the folding transition of apo-SAM-II riboswitch. Average free energy profiles of folding transition at different [Mg²⁺] show significant stability difference between the ligand-free partially closed (PC) and the partially open (PO) minima (ΔG_PC-PO); and the unfolded (U) and the extended open (O) state minima (ΔG_U-O). In the inset ΔG_PC-PO and ΔG_U-O are plotted together as a function of [Mg²⁺]. Both sigmoid curves follow the same transition midpoint, Mg_1/2 ≈6.0 mM, as found in the SEC profile.