(A) Schematic representation of riboswitch and control constructs. The sequence of the theophylline aptamer is labeled in red, the hairpin loop region in cyan (taken from a transcription-regulating riboswitch construct [Wachsmuth et al. 2013; Ender et al. 2021]), and the complementary region forming the sequester hairpin in blue. The tRNA element is indicated in green (suppressor tRNATyr supF from E. coli). Base positions are numbered relative to the cleavage site and according to Sprinzl et al. (1998). The upstream sequence of the tRNA, represented as N stretch, is replaced by the respective 3′-part of each construct. Upon ligand binding (black oval), the riboswitch refolds, and the 5′-leader is no longer masked in the sequester hairpin and is accessible for cleavage. The natural leader sequence of the control construct nat-supF is indicated in gray. (B) In vitro analysis of riboswitch-controlled tRNA maturation. Cleavage of RP-RS A (central panel) and RP-RS C (right panel) by PRORP1 (P1), PRORP2 (P2), or E. coli RNase P holoenzyme (Eco) compared to supF with the naturally occurring 5′-leader (nat-supF; left panel) (Kirsebom and Svärd 1992). (C) Quantitation of the produced percentual product amount. Only in the case of RP-RS C, a theophylline-dependent tRNA processing is observed for all tested enzymes. Data are mean ± SD, n = 4. (D) Theophylline-induced fold changes of nat-supF (left), RP-RS A (center), and RP-RS C (right). Results show a low theophylline-dependent response ratio of 1.7- (PRORP1) and 1.5-fold (PRORP2) for RP-RS A and a clear activation for RP-RS C (PRORP1: 2.4-fold; PRORP2: 3.7-fold). Data are mean ± SD, n = 4.
