Fig. 3: Na⁺ selectively triggers riboswitch-mediated transcription read-through and increased gene expression.

a, WT Na⁺ riboswitch model based on the DUF1646 motif representative from the C. acetobutylicum kefB gene. Approximate 3′ termini for the terminated (T) and FL RNA transcripts are indicated. FL carries an additional 60 nucleotides (encircled number) not shown. The genetic reporter construct carries different 3′ nucleotides, including the lacZ gene. Additional annotations are as describe for Figs. 1 and 2. b, Top, representative PAGE analysis of in vitro transcription termination assays. Bands corresponding to the T and FL RNA products as defined in a are indicated. Lane M (marker) was loaded with a transcript corresponding to FL RNA. Bottom, a plot of the fraction of FL transcripts versus ion supplementation for various constructs. Data points (n = 3) corresponding to the PAGE autoradiogram depicted are coloured the same as the annotation panels. Open and black-filled data points were derived from two additional replicate assays (autoradiograms not shown). Solid lines represent average values for each condition, and the dashed line represents the average value for the WT construct with no Na⁺ supplementation. All transcription reactions include at least 4 mM Na⁺ due to its presence in the standard transcription reaction buffer, and this is not reflected in the ion identities and concentrations presented in the graphic. c, Representative reporter assays using surrogate B. subtilis cells carrying the riboswitch–reporter construct depicted in a. Cells were cultured in low-sodium LB media at the indicated pH value and containing roughly 15 mM Na⁺ and X-gal, which was used either without alteration (–) or was supplemented with 150 mM NaCl (Extended Data Fig. 5 and Supplementary Table 1). See Methods for additional details. d, Representative reporter assays conducted as described in c using WT or M4 riboswitch reporters cultured in low-sodium LB medium at pH 9, used without alteration (–) or supplementation with the monovalent ions indicated (Extended Data Fig. 5 and Supplementary Table 1). Lower Li⁺ concentration was used to avoid strong growth inhibition.

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