The protein-DNA contcacts in RutR-carAB operator complexes

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric tran- scription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high- resolution contact map of RutRﴗcarP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specifi- city of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutRﴖoperator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR. Finally, we test the validity of this model with site-directed mutagenesis of the helix–turn–helix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins.

Jaar van publicatie:2010

Toegankelijkheid:Open