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

Author:

Dimas Rey P1,Jordan Benjamin R12,Jiang Xian-Li3,Martini Catherine1,Glavy Joseph S2,Patterson Dustin P4,Morcos Faruck356ORCID,Chan Clement T Y14ORCID

Affiliation:

1. Department of Biology, The University of Texas at Tyler, Tyler, TX 75799, USA

2. Department of Pharmaceutical Sciences, Fisch College of Pharmacy, The University of Texas at Tyler, Tyler, TX 75799, USA

3. Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75080, USA

4. Department of Chemistry and Biochemistry, The University of Texas at Tyler, Tyler, TX 75799, USA

5. Department of Bioengineering, The University of Texas at Dallas, Richardson, TX 75080, USA

6. Center for Systems Biology, The University of Texas at Dallas, Richardson, TX 75080, USA

Abstract

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.

Funder

National Science Foundation

National Institutes of Health

UT Tyler Internal Research Grants program

The University of Texas System Rising STARs Program

Welch Foundation

The School of Natural Sciences and Mathematics

Publisher

Oxford University Press (OUP)

Subject

Genetics

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