The protein-DNA contacts in RutR•carAB operator complexes

Nucleic Acids Res. 2010 Oct;38(18):6286-300. doi: 10.1093/nar/gkq385. Epub 2010 May 14.

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 RutR•carP1 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 RutR·operator 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Carbon-Nitrogen Ligases / genetics
  • DNA, Bacterial / chemistry*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Ligands
  • Models, Molecular
  • Nucleic Acid Conformation
  • Operator Regions, Genetic*
  • Protein Structure, Tertiary
  • Thymine / metabolism
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Uracil / metabolism

Substances

  • DNA, Bacterial
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Ligands
  • RutR protein, E coli
  • Transcription Factors
  • Uracil
  • Carbon-Nitrogen Ligases
  • Thymine