Supplementary Figure 3: Transcription antitermination by the B. cereus crcB fluoride riboswitch and mutants.

(a) Single-round in vitro transcription (radiolabeled) of the B. cereus crcB fluoride riboswitch. NaF was included at concentrations of 0 mM, 0.001 mM, 0.01 mM, 0.1 mM, 1 mM, and 10 mM. Terminator readthrough increases in response to fluoride. (b) The locations of mutations M18-M23 from Baker et al. (Baker, J.L. et al., Science. 335, 233-5, 2012) within the antiterminated (high fluoride) and terminated (low fluoride) secondary structures (also shown in Fig. 5a). (c) Table of mutant properties. Aptamer folding was determined from cotranscriptional SHAPE-Seq trajectories. Terminator activity was determined from part (d) and is consistent with reporter fusion assays performed in Baker et al. (Baker, J.L. et al., Science. 335, 233-5, 2012). (d) Single-round in vitro transcription (unlabeled) of the mutants shown in (b) in the presence and absence of 10 mM NaF. Transcription reactions were performed in conditions matching those used for cotranscriptional SHAPE-seq. Lane M is the RNA Century Marker (Ambion) with 100 nt and 200 nt bands shown. Templates with a functional terminator (wt, M18, M22, and M23) show a higher basal level of terminator readthrough in cotranscriptional SHAPE-Seq conditions than in conditions used for radiolabeling in (a), which contain a 10-fold lower UTP concentration. Both the wt riboswitch and the mutants follow previously described trends (Baker, J.L. et al., Science. 335, 233-5, 2012). Mutations that disrupt aptamer formation but allow terminator formation (M18 and M22) terminate at comparable efficiencies to wt without fluoride, regardless of fluoride concentration. Mutations that disrupt the terminator hairpin (M19, M20, and M21) do not terminate regardless of fluoride concentration. The combination of the M18, M19, and M20 mutations in M23 restores both aptamer and terminator hairpin base pairing and behaves as the wt riboswitch. Results shown are n=1.