A strategy for dissecting the kinetics of transcription repression mechanisms

Promoters in Escherichia coli include an 'OFF' state, during which transcription is halted. Here, we propose a novel empirical method for assessing the time-length spent by promoters in this state. It relies on direct measurements of RNA production kinetics at the single molecule level at different induction levels, followed by an estimation of the RNA production rate under infinite induction, which is then compared to this rate under real, maximum induction. We apply it to the LacO301 promoter and infer that, under full induction, on average, 15% of the time between successful transcription events is spent in the OFF state. We verify this result by comparing the kinetics of a mutant strain lacking repressor molecules with that of the inferred rate under infinite induction. We expect this strategy of dissecting the kinetics of transcription repression to be applicable to a wide number of promoters in E. coli.