Metal ion-induced folding of the Arabidopsis and E. coli TPP riboswitch k-junctions analyzed by FRET. (A) Schematic of the constructs used for FRET analysis of k-junctions. The T-helix has been replaced by a 4 bp stem–loop, and the C- and NC-helices have been extended and labeled with 5′ fluorescein (F) and cyanine-3 (C), respectively. The Arabidopsis and E. coli k-junctions have been titrated with metal ions, and FRET efficiency (EFRET) measured and plotted as a function of ion concentration. The data are fitted to a simple two-state binding model (lines). (B) Titration of the Arabidopsis k-junction with Mg2+ ions. Filled circles: natural sequence Arabidopsis k-junction. Open circles: Arabidopsis k-junction with L1H substitution. This removes the hydroxyl group proposed to donate a hydrogen bond to A1n N1. (C) Titration of the E. coli k-junction with Mg2+ ions. Filled circles: natural sequence E. coli k-junction. Open circles: E. coli k-junction with L1H substitution. (D) Titration of the Arabidopsis k-junction with Na+ ions. (E) Titration of the E. coli k-junction with Na+ ions.
