Short talk:
Post-translational lysine acetylation regulates the transcriptional regulator RutR as shown by using a structural biological and synthetic biological approach

Magdalena Kremer1, Sabrina Schulze1, Nadja Eisenbruch1, Robert Vogt1, Leona Berndt1, Britta Girbardt1, Felix Nagel2, Mihaela Delcea2, Michael Lammers1

1University of Greifswald, Institute of Biochemistry, Synthetic and Structural Biochemistry, Greifswald, Germany,
2University of Greifswald, Institute of Biochemistry, Biophysical Chemistry, Greifswald, Germany

Bacteria live in rapidly changing environments with consequences on nutrient availability. They precisely sense the cellular metabolic state and translate this in altered protein functionalities. The TetR-family member RutR (pyrimidine utilization repressor) is a transcriptional repressor for the rutA-G operon involved in pyrimidine breakdown and it activates transcription of the carAB operon involved in pyrimidine biosynthesis. RutR is structurally composed of an N-terminal DNA-binding domain (DBD) and of a C-terminal ligand-binding domain. It is lysine acetylated at distinct sites in the DBD and LBD. How lysine acetylation affects RutR function is not known. Applying the genetic code expansion concept (GCEC) we produced site-specifically lysine- acetylated RutR proteins suitable for biophysical studies including X-ray crystallography. We solved a crystal structure of K52-acetylated RutR showing how K52-acetylation switches-off RutR DNA-binding exerting a steric and electrostatic mechanism. The interactions of acetylated RutR-variants with carAB and rutAB dsDNA operator sequences were analysed thermodynamically ITC and by EMSAs. We applied the GCEC in E. coli in vivo showing that RutR acetylation modulates its transcriptional regulator activity. RutR acetylation is catalysed enzymatically by E. coli lysine acetyltransferases and non-enzymatically by acetyl-phosphate. The sirtuin deacetylase CobB can revert RutR acetylation at all functionally important sites. We present a model explaining how prokaryotes apply lysine acetylation of transcriptional regulators as sensors of the cellular metabolic state directly adjusting gene expression allowing to adjust to changing environmental conditions.


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