  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.
