Engineered Stop and Go T7 RNA Polymerases.

Abstract

Precise, stringent, post-translational activation of enzymes is essential for many synthetic biology applications. For example, even a few intracellular molecules of unregulated T7 RNA polymerase can result in growth cessation in a bacterium. We sought to mimic the properties of natural enzymes, where activity is regulated ubiquitously by endogenous metabolites. Here we demonstrate that full-length, single subunit T7-derived RNA polymerases (T7 RNAP) can be activated by physiologically relevant concentrations of indoles. We used rational design and directed evolution to identify T7 RNAP variants with minimal transcriptional activity in the absence of indole, and a 29-fold increase in activity with an EC₅₀ of 344 μM. Indoles control T7-dependent gene expression exogenously, endogenously, and between cells. We also demonstrate indole-dependent bacteriophage viability and propagation in trans. Specificity of different indoles, T7 promoter specificities, and portability to different bacteria are shown. Our ligand activated RNA polymerases (LARPs) represent a new chemically inducible "stop and go" platform immediately deployable for novel synthetic biology applications, including for modulation of synthetic cocultures.