Fig. 4.

Kinetic assessment of ligand-induced preQ₁ riboswitch folding. (A) Schematics of secondary structure rearrangement of A33Ap labeled variants (34 nt, 61 nt, 70 nt). (B) Location of A33Ap (red) in the aptamer/preQ₁ complex (coordinates based on the crystal structure from Ferre d’Amaré and coworkers of a homologous preQ₁ aptamer from B. subtilis (13); representation of RNA in cartoon and of ligand (preQ₁) in spheres. (C) Determination of apparent K_D value. Fluorescence changes upon titration of A33Ap 70 nt labeled variant with preQ₁. Normalized Ap fluorescence intensity plotted as a function of preQ₁ concentrations. Changes in fluorescence (F-F₀) were normalized to the maximum fluorescence measured in the absence of preQ₁. The graph shows the best fit to a single-site binding model (SI Text). The inset shows fluorescence emission spectra (λ_ex = 308 nm) from 330–480 nm for each preQ₁ concentration; c_RNA = 0.5 μM, 50 mM KMOPS, 100 mM KCl, 2 mM MgCl₂, pH 7.0, at 298 K. (D) Representative fluorescence response of the A33Ap 70 nt variant upon preQ₁ addition; conditions: same as (C) and c_preQ1 = 2.0 μM; mixing was performed with a stopped-flow apparatus. (E) Plot of observed rate k^′ versus ligand concentration for the three different A33Ap variants as indicated. Observed rates were determined under pseudo-first-order conditions from three independent stopped-flow measurements. The slope of the plot yields the rate constant k (see Table 1).