The natural k-junction can be functionally substituted by the DUF-3268 k-junction sequence in the E. coli TPP riboswitch. (A) Schematic showing a chimeric TPP riboswitch with the DUF-3268 k-junction (boxed). The position of the A1nU substitution is shown. (B) Analysis of the ligand-induced global conformation of the TPP riboswitch variants by polyacrylamide gel electrophoresis. The natural sequence E. coli TPP riboswitch and the DUF-3268 chimera with and without the A1nU substitution were electrophoresed in a 10% polyacrylamide gel after incubation with no ligand, 0.1 or 1 mM TPP. (C–E) ITC of the three riboswitch variants. A solution of TPP was titrated into the riboswitch solutions, and the heat evolved was measured as the power required to maintain zero temperature difference with a reference cell. Integration over time gives the heat required to maintain thermal equilibrium between cells. In each case the upper panel shows the raw data for sequential injections of 2 µL volumes (following an initial injection of 0.4 µL) of a 250 µM solution of TPP into 200 µL of a 30 µM RNA solution in 40 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2. This represents the differential of the total heat (i.e., enthalpy ΔH° under conditions of constant pressure) for each TPP concentration. The lower panels show the integrated heat data fitted (where possible) to a single-site binding model. The thermodynamic parameters calculated are summarized in Supplemental Table S2.
