Fig. 5: Application of NPOM for the study of the C158 pause in the E. coli thiC riboswitch.

a The thiC riboswitch contains two consecutive transcriptional pauses. Due to its upstream location, ECs escaping the A138 pause may artificially increase the half-life of the C158 pause. In the context of a A138 hairpin mutant, ECs escape the A138 pause at a faster rate, therefore artificially decreasing the C158 pause half-life. Using an NPOM-containing template to synchronize EC at the C158 pause site, the measured C158 pause half-life is expected to strictly represent the rate of pause escape, which should not be affected by the half-life of the A138 pause. b Observed half-life values of pauses A138 and C158 in the context of the wild-type and mutant hairpin constructs. Experiments were performed with and without NPOM when monitoring the C158 pause. The average values of independent experiments with standard deviations are shown. c Transcription kinetics experiment monitoring the half-life of the C158 pause in the context of the hairpin mutant. ECs were synchronized at the C158 pause site using a NPOM roadblock. NPOM removal was performed in the absence of NTPs. The experiments were performed at least two times and variations were less than 10%.