The proteinDNA contacts in RutR carAB operator complexes

Author: Nguyen Le Minh Phu  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.38, Iss.18, 2010-10, pp. : 6286-6300

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

Abstract

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription 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 RutRcarP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity 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 RutRoperator 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 helixturnhelix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins.