Extended Data Fig. 3: Transcriptional regulation of LIRAs and comparison to toehold switches.

a, Structure and sequences of selected toehold switches used for testing translational leakage in Fig. 2c. b, ON-state GFP fluorescence of toehold switches and LIRAs tested for translational leakage in Fig. 2c. n = 3 biological replicates, bars represent the geometric mean ± s.d. c, Schematic of the putative transcriptional regulation mechanism of LIRAs. The strong hairpin structure of the LIRA can cause early transcriptional termination for some transcripts. In the presence of the input RNA, binding of the input prevents formation of the hairpin structure and promotes transcription of the full-length mRNA. d, Minimum free energy (MFE) of the T7 terminator and the hairpin structures of the toehold Switches and LIRAs from Fig. 2c. Toehold switches have a weaker secondary structure than the T7 terminator, which makes them unlikely candidates for transcriptional termination. LIRAs possess a longer hairpin structure and have a stronger secondary structure than the T7 terminator. The stronger hairpin structure is expected to encourage transcriptional termination, albeit at a lower rate than natural terminators that feature 3’ U-tracts and short loop domains to promote faster hairpin formation.