Probing the dependence of transcription factor regulatory modes on promoter features

Transcription Factors (TFs) are often classified as activators or repressors, yet these context-dependent labels are inadequate to predict quantitative profiles that emerge across different promoters. The regulatory interplay between a TFs function and promoter features can be complex due to the lack of systematic genetic control in the natural cellular environment. To address this, we use a library of E. coli strains with precise control of TF copy number. We measure the quantitative regulatory input-output function of 90 TFs on synthetic promoters that isolate the contributions of TF binding sequence, location, and basal promoter strength to gene expression, uncovering TF specific regulatory principles. We infer that many of these TFs function by stabilizing RNA polymerase at the promoter, a property we see for both activating and repressing TFs. We develop a thermodynamic model that predicts stabilizing TFs have a specific quantitative relationship with promoters of differential strength. We test this prediction using synthetic promoters spanning over 100-fold range in basal expression levels and confirm that stronger promoters have lower fold-change for stabilizing TFs, whereas non-stabilizing TFs do not exhibit this relationship, indicating a conserved mechanism of transcription control across distinct TFs. This work demonstrates that understanding the intrinsic mechanisms of TF function is central to decoding the relationship between sequence and gene expression.