Computation-guided redesign of promoter specificity of a bacterial RNA polymerase

Abstract

ABSTRACTThe ability to regulate circuits and pathways is central to cellular control. The existing toolkit is predominantly comprised of local transcription regulators that are unsuitable for exerting control at a global genome-wide scale. Bacterial sigma factors are ideal global regulators as they direct the RNA polymerase to thousands of transcription sites. Here, we redesigned the promoter specificity of theE. colihousekeeping sigma factor, sigma-70, toward five orthogonal promoter targets not recognized by the native sigma-70. These orthogonal sigma-70s were engineered by screening a pooled library of computationally-guided designs customized toward each promoter target. A combination of conserved interactions with the DNA backbone and target-specific interactions facilitate new promoter recognition. Activity of the top performing redesigned sigma-70s varied across the promoter targets and ranged from 17% to 77% of native sigma-70 on its canonical active promoter. These orthogonal sigma factors represent a new suite of regulators for global transcriptional control.