Single-base substitutions switch RNA tertiary allostery between positive and negative coupling.

Riboswitches regulate gene expression in response to ligand binding, remodeling their secondary structure to terminate transcription or block translation. This innate switching has driven efforts to repurpose riboswitches as synthetic control elements. Although promising, tuning riboswitch responses to new ligands and functions has proven difficult. Large RNAs such as the ribosome and RNase P exhibit an alternative switching mode via the docking and undocking of tertiary contacts. These transitions are buried deep within the machines, and there is no minimal system to study them in isolation. To overcome this limitation, we built a minimal 3D-structure ligand-inducible switch. We started with a previously designed nanostructure containing an ATP-aptamer and a tetraloop/tetraloop receptor (TL/TLR) tertiary contact. DMS-MaPseq screening of 3,375 mutants of the two linking motifs, a kink-turn and a 4-1 junction, identified 237 variants in which AMP binding undocked the TL/TLR and 120 in which it drove docking. Switching arose almost entirely from mutations in the conserved sheared G·A base pairs of the kink-turn. Fitting four representative constructs' AMP titrations to a linked-equilibrium model yielded coupling free energies spanning 3.7 kcal/mol, and magnesium titrations with and without AMP independently confirmed these couplings while revealing a three-state docking pathway in mutants lacking wild-type coupling. Single nucleotides inserted into the kink-turn motif adjust the sign and strength of coupling without redesigning the secondary structure. This simple system thus provides a platform both as a model for tertiary switching in other large RNA machines and as a foundation for designing synthetic control elements based on tertiary interactions.