1. Engineering DNA recognition and allosteric response properties of TetR family proteins by using a module-swapping strategy
- Author
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Dustin P. Patterson, Xian-Li Jiang, Joseph S. Glavy, Clement T Y Chan, Benjamin R Jordan, Rey P Dimas, Catherine Martini, and Faruck Morcos
- Subjects
Models, Molecular ,Protein Conformation, alpha-Helical ,Recombinant Fusion Proteins ,Genetic Vectors ,Regulator ,Gene Expression ,Computational biology ,Lac repressor ,Biology ,Crystallography, X-Ray ,Protein Engineering ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Allosteric Regulation ,Genetics ,Escherichia coli ,TetR ,Protein Interaction Domains and Motifs ,Cloning, Molecular ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Base Sequence ,business.industry ,Escherichia coli Proteins ,Rational design ,Protein engineering ,DNA ,Modular design ,biochemical phenomena, metabolism, and nutrition ,Repressor Proteins ,Kinetics ,chemistry ,Mutation ,Nucleic Acid Conformation ,UniProt ,business ,Synthetic Biology and Bioengineering ,Sequence Alignment ,030217 neurology & neurosurgery ,Protein Binding ,Transcription Factors - Abstract
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.
- Published
- 2019