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. A mechanistic understanding of how different regulatory sequences shape TF function is challenging due to the lack of systematic genetic control in endogenous genes. To address this, we use a library of Escherichia coli strains with precise control of TF copy number, measuring 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. We interpret the measured regulation of these TFs using a thermodynamic model of gene expression and uncover stabilization of RNA polymerase as a pervasive regulatory mechanism, common to both activating and repressing TFs. This property suggests ways to tune the dynamic range of gene expression through the interplay of stabilizing TF function and RNA polymerase basal occupancy, a phenomenon we confirm by measuring fold change for stabilizing TFs across synthetic promoter sequences spanning over 100-fold basal expression. Our work deconstructs TF function at a mechanistic level, providing foundational principles on how gene expression is realized across different promoter contexts, with implications for decoding the relationship between sequence and gene expression.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.