Fig. 1: Cbe pfl terminator loop length and sequence tune riboswitch sensitivity.
From: RNA folding kinetics control riboswitch sensitivity in vivo
a Secondary structure schematic of the Cbe pfl riboswitch immediately preceding EP nucleation. The polyA terminator loop (purple) closes to allow the invader domain (gold) to initiate strand displacement of the P3 stemloop (red). Ligand binding kinetically competes with this process to determine the riboswitch genetic decision. b EC50 values for riboswitch variants with homopolymeric loops of varying lengths. c Normalized dose response curves of riboswitch variants with varying polyA loop lengths. d Vfold3D models of P3 with un-nucleated 7nt polyA or polyU terminator loops. PolyA (purple, left) exhibits regular stacking that separates the invader nucleotides (gold) from the substrate nucleotides (red) in the P3 stem, whereas polyU (teal) exhibits no regular order. e EC50 values for riboswitch variants with length 7 polyA loops in which one residue has been mutated to either C (red) or G (blue). Nucleotide coordinates are relative to the 3’ end of the loop. Solid horizontal line indicates EC50 value for WT polyA, with dashed horizontal lines indicating the standard error. Cartoon insets illustrate how A→C mutations (top) will disrupt local stacking, while A→G mutations allow local stacking interactions (bottom). f Fold change of the mutants in (e). Solid horizontal line indicates fold change of WT polyA, with dashed horizontal lines indicating the standard error. Data in panel (c) indicates normalized dose response curves over n = 9 replicates, with points indicating mean and error bars indicating standard deviation. Dose response curves were normalized by dividing all data points for each mutant by (Fmax  –  Fmin) of the fitted curve, with standard deviation calculated from the 9 normalized replicate values for each concentration. Data in panels (b), (e), and (f) are determined as described in Methods, from dose response curves including n = 9 biological replicates at each of 8 concentrations. Error bars in (b), (e), and (f) indicate standard error of the fit parameter. Source data are provided as a Source Data file.
