Figure 1: NMR and SHAPE analysis of tP5abc.

(a) Structure of tP5abc with and without Mg²⁺. Magnesium ions identified in the X-ray structure (PDB ID: 1GID) are represented as orange spheres. Non-Watson–Crick base pairs in tP5abc^Nat are represented by Leontis/Westhof symbols⁶⁶. Five base pairs are removed from P5b relative to the natural sequence. Tertiary interactions are indicated using dashed lines: U168 stacks on A183; A186 forms H-bonds with G181, C137 and G164. Residues undergoing change in secondary structure are shown in red. Residues with imino resonances that undergo changes or disappear on addition of Mg²⁺ are circled in black. (b) Overlay of 2D ¹H-¹⁵N HSQC spectra of 1 mM tP5abc in the absence of Mg²⁺ (black) and 0.1 mM tP5abc in the presence of 7.5 mM Mg²⁺ (red) in 10 mM sodium phosphate and 0.01 mM EDTA, pH 6.4, at 10 °C. MgCl₂ was directly added to the NMR sample. Resonances belonging to tP5abc^Nat are labelled with asterisks based on the previous assignments²⁶. The resonances labelled with ‘+’ represent alternative states for residues G131 and G191 due to minor N+1 transcripts. (c) Differential SHAPE reactivity of 0.2 μM WT P5abc in the presence of 10 mM Mg²⁺ relative to no Mg²⁺ conducted in 10 mM sodium phosphate and 0.01 mM EDTA, pH 6.4, at 10 °C. Red (blue) represents more (less) reactivity of one state relative to the other. The extended P5abc construct is shown in Supplementary Fig. 2a.