RNase H assays of FL ribB nascent riboswitch transcripts. (A) Schematic representing the ribB riboswitch in the FMN-bound OFF conformation. The P1 and anti-P1 stems are shown in green and orange, respectively. The RNase H probes 137, 184, and 218 are shown in purple. (B–D) RNase H probing assays performed on FL (282 nt) nascent ribB riboswitches. Reactions were done as a function of the FMN concentration using probes 137 (B), 218 (C), and 184 (D). Cleavage assays were performed using a range of 100 pM to 500 µM FMN. FL and cleaved products (P) are indicated on the right of the gels. For each reaction, the Kswitch value was determined by fitting the data to a two-state model. The experiments were performed in triplicate, and the most representative data are shown.
