Steinmetzger, C.; Karlsson, H.; Fontana, C.; Galindo Riera, N.; Riad, M.; Kosek, D.; Schlagnitweit, J.; Steiner, E.; Friebe Sandoz, S.; Andersson, E. R.; Petzold, K.

A-minor motifs consist of adenosines docking into adjacent RNA minor grooves, are the most prevalent 3D interaction stabilizing RNA structures, and widely considered as being static. NMR spectroscopy reveals a secondary structure equilibrium of these motifs between engaged and disengaged states, which we term the A-minor switch. A switch in E.coli ribosome helix 44 consists of a sparsely populated, transient single-nucleotide register shift that sequesters adenosines from their 3D structural A-minor contacts. Mutational trapping of the NMR-defined, A-minor-engaged ground and -disengaged excited state, combined with cryo-electron microscopy, visualizes this dynamic switch mechanism. Additionally, A-minor switches were identified using secondary structure ensemble analysis and mutational trapping was found to impair bacterial growth, directly linking RNA dynamics and function. Because A-minor motifs are widespread in structured RNAs, these findings establish A-minor switches as a general regulatory layer between secondary structure dynamics and tertiary contacts, exposing a new therapeutic target class.