Figure 4.

Serial riboswitch constructs. (A) Schematic representation of tandem and tridem constructs upstream of the reporter gene (β-galactosidase or GFP), in 5′ to 3′ orientation. All transcripts start with a 3 nt-leader upstream of the first aptamer domain. For cloning reasons, several constructs carry a 6 nt-gap between the repeats, indicated by a black line. If no line is present, riboswitches directly follow each other. When indicated, an inert 19 nt region of the pBAD multiple cloning site (see ref.¹²) is inserted between riboswitch and Shine-Dalgarno sequence (RSshift constructs). The same arrangement was chosen for constructs with GFP reporter. (B) β-galactosidase activity of constructs with up to 3 serial riboswitch copies. While in the RS10 constructs the response ratio is increasing with additional riboswitch copies, the RS10–10–10shift construct shows a drop in activation compared to the corresponding tandem. (C) β-galactosidase activity as a function of theophylline concentration for the constructs RS10 (red), RS10–10 (orange) and RS10–10–10 (green). The linear regression coefficient R² (colored accordingly) indicates that the linearity increases with the number of riboswitches. (D) β-galactosidase activity of tandem combinations of RS10 with RS1 or RS3, respectively. The approximate maximal activation ratio for all constructs is given as a number above each column set.