Abstract
Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work we characterize the PcaR TF belonging to the IclR family. We present in vitro functional analysis of PcaR’s ligand profile and construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this TF. This work provides key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors.
Highlights
PcaR is an IclR family transcription regulator responsive to dicarboxylic acids
PcaR was established as an in vivo biosensor in yeast
Crystal structure of PcaR in the apo form was solved
Crystal structure with PcaR in complex with succinate was solved
Sequence alignments unveil ligand-binding positions in the IclR family
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- TF
- transcription factor
- DSF
- differential scanning fluorimetry
- DBD
- DNA binding domain
- LBD
- ligand binding domain
- PDB
- protein data bank
- HTH
- helix-turn-helix
- DSF
- differential scanning fluorimetry
- RNAP
- RNA polymerase
