Decoding and reengineering the promoter specificity of T7-like RNA polymerases based on phage genome sequences.

Abstract

The single subunit RNA polymerases (ssRNAPs) of bacteriophages are highly interesting targets for the prediction and engineering of specific protein-DNA interactions. Despite extensive existing studies focusing on particular ssRNAPs such as the T7 RNAP, few rules governing the protein-DNA sequence covariations across diverse ssRNAPs and their cognate promoters are clearly known. Here, aiming to reveal such rules, we comprehensively mined promoters of various categories of ssRNAPs from phage genomes. For T7-like RNAPs, direct coupling analyses of the predicted set of RNAP-promoter pairs revealed that the interaction specificity was dominantly encoded by the amino acid and nucleotide residues at only a few key positions. The covariations between the amino acid and the nucleotide residues at these positions were summarized into a sparsely connected network. Using experimentally verified connections in this network, we designed a set of orthogonal T7 RNAP-promoter variants that showed more stringent orthogonality than previously reported sets. We further designed and experimentally verified variants with novel interactions. These results provided guidance for engineering novel RNAP-promoter pairs for synthetic biology or other applications. Our study also demonstrated the use of comprehensive genome mining in combination with sequence covariation analysis in the prediction and engineering of specific protein-DNA interactions.