Purified Can RNA was labeled with 32P-γ-ATP via T4 Polynucleotide Kinase. The construct sequence was: 5′-UGUGGUUCGCAACCAUCCCACAUAAAAAAACU AG GAGGAUUCACACAUG UAGACAAAAAAUAUCGUCACAAA. Here, the WT Can riboswitch (underlined) is followed by the native sequence through the start codon (bold). A stop codon (italics) was placed immediately downstream. Radiolabeled RNA was purified by passing through a G-25 spin column (Cytiva, MA). Translation initiation and RelE cleavage were performed as described (38 (link)). Concentrations of preQ1 ranged from 0 to 500 μM, and translation initiation reactions were incubated at 37 °C for 30 min prior to RelE cleavage. Full-length and cleaved species were separated on a 10% denaturing polyacrylamide gel, visualized with a Typhoon imager (Cytiva), and analyzed with ImageQuant (Cytiva).
Band intensities were analyzed under the assumption that the concentration of the labeled RNA is much less than the K1/2. The data were fit in Prism 9 (GraphPad, Inc.) with the following equation:
where X is the concentration of ligand; Y is the percent cleaved; K is the K1/2; Ymin is the minimum percent cleaved; amplitude is the difference between Ymax and Ymin; and n is the Hill coefficient.
Band intensities were analyzed under the assumption that the concentration of the labeled RNA is much less than the K1/2. The data were fit in Prism 9 (GraphPad, Inc.) with the following equation:
where X is the concentration of ligand; Y is the percent cleaved; K is the K1/2; Ymin is the minimum percent cleaved; amplitude is the difference between Ymax and Ymin; and n is the Hill coefficient.
