Secondary structure analysis by in-line probing. (A) Constructed secondary structures of ON and OFF states of riboswitches RP-RS A-P and RP-RS C-P fused to tRNAPyl. The theophylline-binding aptamer is presented in red, the hairpin loop region in cyan, the designed 3′-part of the sequester hairpin in blue, and tRNAPyl in purple. The anticodon is shown in black. Bars in green, purple, orange, brown, and cyan represent regions identified as single-stranded in the corresponding structures. In the ON state of RP-RS A-h, additional possible interactions of the blue sequester part with the aptamer base are indicated by dashed lines and a question mark. (B) In-line probing patterns of riboswitch regions in RP-RS A-P and RP-RS C-P. Black horizontal lines in the two panels indicate a cut of the gel picture due to its length. Both constructs show a ligand-dependent opening of the sequester hairpin. In the uninduced state (absence of theophylline indicated by “−”), RP-RS A-P shows fraying of the basal three base pairs of the hairpin, supporting previous data on this construct (Ender et al. 2021). (n) Negative control (transcript without incubation), (T1) partial digest with RNase T1, (OH−) alkaline hydrolysis, (−/+) incubation in in-line probing buffer for 40 h in the absence (−) or presence (+) of theophylline.
