Cryo-EM reveals remodeling of a tandem riboswitch at 2.9 Å resolution.

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 a combination of molecular dynamics and cryo-electron microscopy to expound the flexible nature of the glycine riboswitch tandem aptamers and characterize diMerent structural populations. We find that Mg partially stabilizes the fully folded state, resulting in one-third of the particles adopting a unique "walking man" conformation, consisting of a rigidified core and two dynamic helices, and two-thirds adopting distinct, partially folded states. Glycine interactions double the relative population of fully folded particles by stabilizing a conserved inter-aptamer Hoogsteen base pair, enabling our capture of a 2.9 Å structure for this RNA-only system. The population data show that glycine and Mg operate synergistically: glycine enhances Mg occupancy, while Mg drives glycine specificity. Our findings indicate that cryo-electron microscopy oMers a promising avenue to characterize RNA folding ensembles.