Fig. 2.
Secondary structure model for the 61 nt riboswitch construct in the absence of preQ1. (A) Probing for mapping a structural rearrangement in the expression platform in vitro by lead(II)-acetate in the absence (-) and presence (+) of a 4-fold excess of preQ1. C designates control reactions without and with preQ1. T1 and H designate RNase T1 and alkaline ladders, respectively. A specific and prominent Pb2+ cleavage site is evident between C45 and A46 (indicated by a red rectangle in dashed line). Conditions: cRNA = 2.5 μM; cpreQ1 = 12.5 μM; buffer: 50 mM KMOPS, 100 mM KCl, 2 mM Mg2+, pH 7.0, 298 K; lead(II)-acetate: 1 mM. (B) Bistable secondary structure model for the riboswitch in the absence of preQ1. (C) Evidence for a bistable segment (21 nt RNA) in the expression platform by comparative imino proton 1H NMR spectra. (D) Same bistable segment [as in (C)] labeled with noninvasive 5-F uridine and verification of 45∶55 equilibrium between fold A and B by 19F NMR spectroscopy [assignment based on short reference sequences as in (C)]. Conditions: cRNA = 0.5 mM, 25 mM sodium arsenate buffer, H2O/D2O = 9/1, pH 6.5, 298 K.