FIGURE 3.

Thermodynamic cycle for S4 binding. (A) Four-state model for S4 titrations contains high-FRET native (N-S4) and low-FRET intermediate (F-S4) S4-bound complexes and corresponding unbound states (N and F). (B) Titrations with S4-Cy5 at different [Mg²⁺]. Lines represent the best least-squares fit to Equation 1. The initial slope is sensitive to β = K_N(1 + K₁). The endpoints are proportional to (E_N + E_F K₂)/(1 + K₂). See Supplemental Material for further details. (C) Ratio of N-S4 and F-S4 complexes, K₂, as a function of [Mg²⁺]. The smooth curve is only intended to guide the eye. (D) Equilibrium dissociation constant for the native complex (K_N, blue diamonds), using K₁ from hydroxyl radical footprinting of free 5′-domain RNA. The calculated dissociation constant for the flipped complex (K_F, red circles) does not increase with Mg²⁺. (E) S4-5′-domain complexes give higher FRET signal in 20 mM MgCl₂ in the presence of both proteins S16 and S20 (green squares) compared to only S16 (blue diamonds) or without any additional protein (pink circles). (F) Titration curves in 4 mM [Mg²⁺]. S4-RNA complexes formed in the presence of proteins S20 and S16 (green) show high FRET compared to S4 alone (pink).