Fig. 3

Precursor tmRNA can act as an RNA sensor for FMN. a Schematic of the B. subtilis tmRNA genomic locus (top) and predicted secondary structures of the long precursor tmRNA, short precursor tmRNA, and mature tmRNA, using the RNAfold program²⁰ (bottom). b RNA footprinting analysis of the long precursor tmRNA, using a SHAPE-like chemical (NAI), in the presence (lane 4) and absence (lane 3) of 100 µM FMN. Also shown are A ladder (lane 1) and unmodified RNA (lane 2). The red bar indicates bases that become more single-stranded in the presence of FMN. c Predicted secondary structure of the tmRNA long precursor using the RNAfold program²⁰. The red bases correspond to the positions marked by the red bar in b. d Average footprinting analysis (n = 3, SAFA) of mature (top), short precursor (middle), and long precursor tmRNA (bottom), in the presence (red) and absence (black) of 100 µM FMN, in the dark. The beige box indicate the region of increased flexibility in the precursor tmRNAs in the presence of FMN. The stars indicate bases that show statistically significant changes with FMN (p ≤ 0.05, Student t-test). e qPCR analysis of the mRNA expression level of precursor tmRNA and mature tmRNA, across six biological replicates, after addition of 100 µM of riboflavin to the growth media of B. subtilis. Fold changes are normalized to the negative control Veg gene. The known B. subtilis FMN riboswitch is used as the positive control. p-values were calculated by Student’s t-test, the error bars indicate standard deviation of the replicates