Figure 5.

Mutational analysis of the α and α′ interaction in the NCU01977 TPP riboswitch. (A) Plot of LUC reporter gene expression for various constructs in the absence (−) or presence (+) of thiamin supplementation. Reporter constructs for Del α (deletion of the α element) and M1 through M6 (nucleotide changes depicted in Figure 4A) are based on the parent construct depicted in Figure 3A. Del G (has a deletion of a conserved G in the junction of P2 and P3 that disrupts TPP binding) and the double mutant Del α/Del G (deletion of α and G) are based on the original WT construct that retains intron 1 and the natural P3 stem. (B) Agarose gel separation of RT-PCR products from WT and all mutant constructs. Products I through IV are similar to those observed in Figure 1B, except that they are ∼200-nt shorter owing to the use of different primers.