FIGURE 6.

The single-nucleotide bulge has a profound effect on thermometer behavior. (A) Representative SHAPE results at 30°C (top) and 42°C (bottom) for the closed control RNA lacking the C bulge (ΔC control). (B) SHAPE results mapped on the predicted secondary structure of the hairpin in the ΔC control RNA. The position of the deleted nucleotide is indicated with an arrow. (C) SHAPE melting curves comparing the melting behavior of the ΔC control RNA with the wild-type thermometer. Solid lines show melting curves for selected nucleotides in the 3′ side of the ΔC control stem (A38, A39, A45, U46, and U47, with numbering as in the wild-type sequence). Each data point is the average of two independent experiments. Error bars are not shown in the interests of clarity (see Supplemental Fig. 3 for representative curves with error). Melting curves from the corresponding nucleotides for the wild-type RNA (from Fig. 3B) are shown with dashed lines. (D) Comparison of the wild-type thermometer at 42°C and the ΔC control at 56°C. Shown are the lowest energy SHAPE-derived secondary structures, with nucleotides color-coded by the average SHAPE values that were incorporated into structure predictions. The C nucleotide that is removed to make the ΔC control is marked; note that its absence precludes formation of the alternate hairpin. SHAPE values used represent the average of two to four experiments.