Fig. 4: Natural ZTP riboswitch EPs use multiple approaches to enhance sensitivity.

a The EPs of 1755 putative ZTP riboswitches were analyzed for the presence of a downstream polyU tract (730/1755), the presence of a consensus P3 stem (695/730), and the presence of an invader domain that pairs with P3 and competes with the pseudoknotted region for base pairs (287/695). These 287 EP candidates were manually classified into 3 categories based on the spacer region between P3 and the invader as indicated in panel (b): loop only, remote toehold, and direct toehold. A complete table of the 287 EP candidates and their classifications is provided in the Source Data file. (c) Secondary structure schematic of a panel of loop only, direct toehold, and remote toehold EPs (color coding as in panels (a) and (b) selected for further functional characterization. Illustrated regions were chimerically inserted in place of the Cbe pfl polyA terminator loop. The Rsp spacer region is a remote toehold because the invader includes a G108∆ deletion, so G108 was deleted from the invader region in the Rsp chimera. A table of the full sequences of these riboswitches and their genomic loci can be found in Table S1. d In vivo fluorescence data for chimeric riboswitches ordered according to spacer length, with Cbe indicating the WT Cbe pfl sequence. (e) EC₅₀ measurements of chimeric ZTP riboswitches shown in (d). f Fold change of chimeric ZTP riboswitches shown in (e). Bars in panel (d) indicate average Fluorescence/OD₆₀₀ values over n = 9 biological replicates. Data in (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 (e) and (f) indicate standard error of the fit parameter. Source data are provided as a Source Data file.