Abstract
SUMMARYGene regulation often results from the action of multiple transcription factors (TFs) acting at a promoter, obscuring the individual regulatory effect of each TF on RNA polymerase (RNAP). Here we measure the fundamental regulatory interactions of TFs in E. coli by designing synthetic target genes that isolate individual TFs’ regulatory effects. Using a thermodynamic model, each TF’s regulatory interactions are decoupled from TF occupancy and interpreted as acting through (de)stabilization of RNAP and (de)acceleration of transcription initiation. We find that the contribution of each mechanism depends on TF identity and binding location; regulation immediately downstream of the promoter is insensitive to TF identity, but the same TFs regulate by distinct mechanisms upstream of the promoter. These two mechanisms are uncoupled and can act coherently, to reinforce the observed regulatory role (activation/repression), or incoherently, wherein the TF regulates two distinct steps with opposing effects.
Highlights
Transcriptional regulation of gene expression is one of the major mechanisms by which cells respond to cues and stimuli
Despite the diverse nature of the transcription factors (TFs) tested, the regulation for all TFs immediately downstream is consistent with a form of repression that is set by the degree of occupancy of the TF at the promoter independent of TF identity
Thermodynamic model for single TF regulation To deconvolve the role of TF copy number, binding affinity, and binding location from the intrinsic regulatory interactions of the TF with polymerase, we use a thermodynamic model of gene expression (Ackers et al, 1982; Kuhlman et al, 2007; Bintu et al, 2005; Kinney et al, 2010; Buchler et al, 2003; Vilar and Leibler, 2003; Garcia et al, 2012), where we consider only a single TF acting on an otherwise unregulated gene
Summary
Transcriptional regulation of gene expression is one of the major mechanisms by which cells respond to cues and stimuli. Measurements of TF function are the result of knocking out the endogenous TF and observing how gene expression changes as a result This serves a purpose in predicting the specific role of that TF on a given gene but offers less predictive power when examining regulation of other genes by that same TF or to different binding sites. Further characterizing the regulatory function of TFs beyond this small subset should provide a more complete toolset for broader synthetic design purposes
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