Structural basis for Mg²⁺-glycine synergy in the tandem glycine riboswitch revealed by cryo-EM

Riboswitches are non-coding RNA sequences that control cellular processes through ligand binding. Conformational heterogeneity is fundamental to riboswitch functionality, yet this same attribute makes structural characterization of these mRNA elements challenging. Here, we use cryo-electron microscopy to characterize different structural populations of the glycine riboswitch tandem aptamers, and molecular dynamics simulations to support the observed ligand orientation in the holo state. We find that Mg²⁺ enables formation of the fully folded "walking man" conformation, which consists of a rigidified core and two dynamic helices, but is insufficient on its own to make this the dominant state. In Mg²⁺ conditions alone, approximately one-third of the picked particles are fully folded, whereas two thirds adopt distinct, partially folded states. Glycine interactions further stabilize the fully folded conformation, doubling the relative population of fully folded particles by stabilizing a conserved inter-aptamer Hoogsteen base pair, enabling our capture of a 2.9 Å-resolution structure for this RNA-only system. The population data show that glycine and Mg²⁺ operate synergistically: glycine enhances Mg²⁺ localization, while Mg²⁺ drives glycine recruitment. Our findings indicate that cryo-electron microscopy offers a promising avenue to characterize RNA folding ensembles.