Abstract
A canonical quantitative view of transcriptional regulation holds that the only role of operator sequence is to set the probability of transcription factor binding, with operator occupancy determining the level of gene expression. In this work, we test this idea by characterizing repression in vivo and the binding of RNA polymerase in vitro in experiments where operators of various sequences were placed either upstream or downstream from the promoter in Escherichia coli. Surprisingly, we find that operators with a weaker binding affinity can yield higher repression levels than stronger operators. Repressor bound to upstream operators modulates promoter escape, and the magnitude of this modulation is not correlated with the repressor-operator binding affinity. This suggests that operator sequences may modulate transcription by altering the nature of the interaction of the bound transcription factor with the transcriptional machinery, implying a new layer of sequence dependence that must be confronted in the quantitative understanding of gene expression.
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► Location of lac repressor binding site determines mechanism of repression ► For upstream binding, stronger binding sites lead to lower levels of repression ► Operator sequence can modulate the interaction between repressor and RNAP
A widely employed assumption is that the unique role of operator sequence is to determine the affinity for transcription factors. In this model, strong repressor binding site leads to a high level of repression. Gelles, Kondev, Phillips, and colleagues show that this assumption does not always hold in the context of the paradigmatic Lac repressor in E. coli. They show that repressor can act on polymerase by modulating the rate of transcription initiation and that this modulation is dependent on the sequence of the operator such that weaker operators lead to higher repression than stronger operators.