Competition between binding sites determines gene expression at low transcription factor concentrations

The response of gene expression to intra-and extra-cellular cues is largely mediated through changes in the activity of transcription factors (TFs), whose sequence specificities are largely known. However, the rules by which promoters decode the amount of active TF into gene expression are not well understood. Here, we measure the activity of 6500 designed promoters at six different levels of TF activity in budding yeast. We observe that maximum promoter activity is determined by TF activity and not by the number of sites. Surprisingly, the addition of an activator-binding site often reduces expression. A thermodynamic model that incorporates competition between neighboring binding sites for a local pool of TF molecules explains this behavior and accurately predicts both absolute expression and the amount by which addition of a site increases or reduces expression. Taken together, our findings support a model in which neighboring binding sites interact competitively when TF is limiting but otherwise act additively\n\nSignificance StatementIn response to intracellular and extracellular signals organisms alter the concentration and activity of transcription factors (TFs), proteins that regulate gene expression. However, the molecular mechanisms that determine the response of a target promoter to changes in the number of active TF molecules are not well understood. By combining mathematical modeling with measurements of TF dose-response curves for thousands of designed promoters, we show that competition for active TF molecules is a major factor in determining gene expression. At low TF concentrations additional activator-binding sites within a promoter can actually reduce expression. Thermodynamic modeling suggests that steric hindrance between neighboring binding sites cannot explain this behavior, but that competition for limiting TF molecules can.