FRET-guided integrative modeling resolves Mg2+-dependent RNA tertiary-contact formation
Tertiary contact interactions between GNRA tetraloops and their receptors are recurrent motifs that stabilize complex RNA folds and in the context of ribosome biogenesis contribute to ribosomal subunit maturation. High-resolution structure determination methods typically yield single structural snapshots, which may not fully describe the conformational collection populated by such contacts in solution. Here, we investigate a ribosomal RNA tertiary contact formed by a GAAA tetraloop and a kissing loop acting as its receptor using single-molecule FRET-guided integrative modeling. We used a minimal RNA model construct in which the tetraloop and its kissing loop receptor are connected by flexible poly(A)-linkers, assembled its bound state from the native ribosomal cryo-EM geometry, and combined restrained and unrestrained all-atom molecular dynamics simulations with in silico FRET and dynamic fluorescence anisotropy predictions to compare with the respective experiments under varying salt conditions. The modeled bound state matches the smFRET distribution only at non-physiologically high Mg2+ concentrations. At 10 mM Mg2+, however, the single-molecule FRET distribution remains heterogeneous and is reproduced by mixtures of bound and unbound conformations, indicating their coexistence under intermediate Mg2+ conditions. Thus, smFRET-guided integrative modeling resolves subpopulations invisible to static high-resolution structures and ensemble FRET, providing a route to validate and refine RNA structural models and conformational ensembles in solution.