Pseudouridine modifications of the neomycin-sensing riboswitch (NSR). (A) Comparison of the chemical structures of uridine (top) and pseudouridine (bottom) and their atomic numbering schema. (R) Ribose. (B) Secondary structure of the WT NSR and nucleotide numbering. The apical loop comprising the U-turn motif (U14, U15, A16) and the closing U13:U18 base pair is highlighted by a blue frame. A17—the only nucleotide of the apical loop in contact with the ligand—is colored in red. Nucleotides that were replaced with pseudouridine in the course of this study are highlighted in bold italic. The position of the U14C mutation is also indicated. (C) Close-up of the three-dimensional structure of the apical loop of the NSR in the complex with ribostamycin. The signature interactions HB1—the hydrogen bond between the U14 imino group and the A17 5′-phosphate group, HB2—the hydrogen bond between the U14 2′-hydroxyl group and the N7 nitrogen of A16 and the lone-pair-π interaction between the Rp oxygen atom of the 5′-phosphate group of A16 and the base of U14 are highlighted. (D) Ribostamycin affinities of the four pseudouridine containing WT NSR variants U14ψ (far left), U13ψ (middle left), U18ψ (middle right) and U13ψ_U18ψ (far right). Shown are the respective ITC thermograms (top) and the resulting binding isotherms (bottom). The respective values of the dissociation constants (KD) which are the averages of three independent experiments are given as insets.
