A set of orthogonal sigma-like transcription factors created by bisecting T7 RNA polymerase

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Thomas Segall-Shapiro, Christopher A. Voigt

Massachusetts Institute of Technology - Synthetic Biology Center, United States

In this work, we describe the creation of a set of transcription factors that loosely mimics the action of prokaryotic core polymerase and sigma factors. In prokaryotes, the core polymerase complex contains the majority of enzymatic function required for transcription, and sigma factors contain DNA binding domains that recruit the polymerase complex to promoters. Since core polymerase is usually limiting, sigma factors compete with each other to bind core and direct transcription proportional to their relative concentrations and binding affinity. We have attempted to recreate this system for use in orthogonal, synthetic genetic programs by bisecting a set of orthogonal phage RNA polymerases. Using a new transposon-based method for generating libraries of split proteins, we split T7 RNA polymerase into two subunits that are active when co-expressed: a larger one that contains much of the catalytic core, and a smaller one that contains the promoter recognition loop. After improving the split protein interactions and activity, we were able to apply this split site to four orthogonal T7 RNA polymerase variants, making a system with one shared ‘core’ protein and four variable ‘sigma-like’ proteins. Both fragments are necessary for transcription, and we have shown that multiple ‘sigma-like’ factors expressed in the same cell compete with each other for a limited pool of the ‘core’ fragment. We envision that this system could prove useful in engineering more complex regulation of synthetic genetic programs in bacteria. The level of the ‘core’ fragment sets the number of polymerases available to the synthetic program as a whole while the relative levels of the ‘sigma-like’ fragments allocates these polymerases among different parts of the program. The levels of ‘core’ could therefore be modulated to avoid stressing or killing the chassis organism without changing relative expression levels within the synthetic system.