Untangling the regulatory role of individual TFs in E. coli

Predicting the effect of individual TFs on gene regulation can be challenging due to their entanglement with promoter specific effects such as TF-TF interactions, or network interactions (feedback, etc). To overcome this limitation, we have developed an approach using systematically designed synthetic gene targets in E. coli to quantify the pure regulatory function of individual TFs and to remove the “context dependent” entanglements of natural promoters. This methodology quantifies the level of regulation exerted on two distinct steps in the transcription process: the stabilization (or destabilization) of RNAP at the promoter and the rate of initiation of RNAP once bound. The magnitude of these two interactions depends on factors such as TF identity, binding position on the promoter and binding site sequence. These two parameters determine an individual TFs net regulatory effect on expression. To demonstrate this, we measured the regulatory characteristics of 30 unrelated TFs from a wide array of TF families and show that natural TFs regulate through a diverse combination of regulatory modes. Crucially, these two mechanisms do not function the same at every promoter indicating that the net regulatory function of a TF is promoter dependent. To demonstrate this principle, we further measured the regulation of these TFs on a library of promoters of different strength and find specific TFs work better on strong promoters rather than weak ones and vice versa. Overall, we find that by distinguishing the regulatory modes of individual TFs we can determine the effectiveness of specific TFs on different promoters.