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

The ability to regulate genetic circuits and metabolic 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 together they direct the RNA polymerase to thousands of transcription sites. Here, we redesigned the promoter specificity of the E. coli housekeeping sigma factor, sigma-70, toward five orthogonal promoter targets not recognized by the native sigma-70. These orthogonal sigma-70 factors were developed by screening a pooled library of computationally designed variants of the -35 DNA recognition helix, each tailored to a specific target promoter. In the redesigned sigma factors new 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.