Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation

Transcription factors (TFs) mediate gene regulation by site specific binding to chromosomal operators. It is commonly assumed that the level of repression is given by the equilibrium binding of a repressor to its operator alone. However, this assumption has not been possible to test in living cells. Here, we have developed a single molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 minutes at the native lac operator in Escherichia coli and that a stronger operator results in slower dissociation rate, but similar association rate. Our findings do not support the simple equilibrium model. The discrepancy can for example be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from TF binding strengths.