Engineering DNA recognition and allosteric response properties of TetR family proteins by using a module-swapping strategy

• Published in: Nucleic acids research 2019-09, Vol.47 (16), p.8913-8925
• Main Authors: Dimas, Rey P, Jordan, Benjamin R, Jiang, Xian-Li, Martini, Catherine, Glavy, Joseph S, Patterson, Dustin P, Morcos, Faruck, Chan, Clement T Y
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Base Sequence; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; DNA - chemistry; DNA - genetics; DNA - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Kinetics; Models, Molecular; Mutation; Nucleic Acid Conformation; Protein Binding; Protein Conformation, alpha-Helical; Protein Engineering; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Sequence Alignment; Synthetic Biology and Bioengineering; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkz666

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.