The tertiary structures of functional RNA molecules remain difficult to decipher. A new generation of automated RNA structure prediction methods may help address these challenges but have not yet been experimentally validated. Here we apply four prediction tools to a remarkable class of double glycine riboswitches that exhibit ligand-binding cooperativity. A novel method (BPPalign), RMdetect, JAR3D, and Rosetta 3D modeling give consistent predictions for a new stem P0 and kink-turn motif. These elements structure the linker between the RNAs double aptamers. Chemical mapping on the F. nucleatum riboswitch with SHAPE, DMS, and CMCT probing, mutate-and-map studies, and mutation/rescue experiments all provide strong evidence for the structured linker. Under solution conditions that separate two glycine binding transitions, disrupting this helix-junction-helix structure gives 120-fold and 6- to 30-fold poorer association constants for the two transitions, corresponding to an overall energetic impact of 4.3 pm 0.5 kcal/mol. Prior biochemical and crystallography studies from several labs did not include this critical element due to over-truncation of the RNA. We argue that several further undiscovered elements are likely to exist in the flanking regions of this and other RNA switches, and automated prediction tools can now play a powerful role in their detection and dissection.
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