Figure 6. Kanamycin B induces an off-pathway conformation of the c-di-GMP-I riboswitch.

(a) The radius of gyration (R_g) of the riboswitch (measured by SAXS) in 0.01 and 1 mM Mg²⁺ (black and grey, respectively) is modulated by various ligands and polyamines. Lines marking the expanded (0.01 mM Mg²⁺, no ligand) and compact (1 mM Mg²⁺,+c-di-GMP) states clearly delineate the range of R_g in these experiments. Spermine, spermidine, c-di-GMP and Mg²⁺ promote significant compaction of the RNA; kanamycin B does not. High-quality SAXS data were recorded for each sample one time. The error in R_g is obtained from the Guinier fit. (b) Isothermal titration calorimetry (ITC) experiments wherein c-di-GMP (500 μM) was titrated into the riboswitch (50 μM) in a buffer containing 2 mM Mg²⁺ and either 0 μM (black) or 200 μM kanamycin B (grey). After each injection of c-di-GMP, the system equilibrates slowly when kanamycin B is present but rapidly in the absence of kanamycin B (black, and inset). The experiment is completed in less than 60 min in the absence of kanamycin B but requires nearly 333 min when kanamycin B is present. ITC experiments were performed in duplicate.