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 been experimentally validated. Here we apply four prediction tools to a paradigmatic class of double glycine riboswitches that exhibit ligand-binding cooperativity. A novel method called bpp_align, 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 map-ping 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 permitting rigorous thermodynamic comparisons, this helix-junction-helix structure has 100-fold effects on glycine binding events and an overall energetic impact of 4.5 ± 0.8 kcal/mol. Prior biochemi-cal and crystallography studies from several labs have missed 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.
Discovery of new RNA stems and motifs from multiple sequence alignments. Demonstrated use to find a 'missing link' in double glycine riboswitches.
This tool provides basic scripts to run the partition executable RNAstructure on hundreds of homolog sequences from RFAM, and to then visualize the results by averaging across these matrices. Examples from purine-binding and double glycine-binding riboswitches are included.