Secondary-structure analysis of the ESE region in the mTot (A), mΔB5 (B), and mΔB6 (C) constructs. The in vitro-transcribed RNAs were enzymatically digested with S1 nuclease and RNases T1 and V1 and reverse transcribed, and the RT products were separated on a sequencing polyacrylamide gel. No enzyme was added to the reaction mixture in lane N. The regions containing the ESE and ESS elements are shown by a vertical line. The upper part of each panel shows the enzymatic analysis of RNA templates of mTot, mΔB5, and mΔB6 constructs. The bottom part reports the cleavages on the optimized secondary-structure predictions. Squares, circles, and triangles indicate S1 nuclease and RNase T1 and V1 cleavage sites, respectively. Black, shaded, and white symbols indicate high, medium, and low cleavage intensities, respectively. The arrow indicates the position of G150, which is present in a stem position in the mTot RNA but shifts to a loop configuration in the mΔB6 mutant.