Ligand binding specificity of RutR, a member of the TetR family of transcription regulators inEscherichia coli

Abstract

RutR is a member of the large family of TetR transcriptional regulators inEscherichiacoli. It was originally discovered as the regulator of therutABCDEFG operon encoding a novel pathway for pyrimidine utilization, but its highest affinity target is the control region of thecarAB operon, encoding carbamoylphosphate synthase. Unlike most other TetR‐like regulators, RutR exerts both positive and negative effects on promoter activity. Furthermore, RutR exhibits a very narrow ligand binding specificity, unlike the broad effector specificity that characterizes some of the well‐studied multidrug resistance regulators of the family. Here we focus on ligand binding and ligand specificity of RutR. We construct single alanine substitution mutants of amino acid residues of the ligand‐binding pocket, study their effect onin vitro DNA binding in absence and presence of potential ligands, and analyse their effect on positive regulation of thecarP1 promoter and negative autoregulationin vivo. Although RutR structures have been determined previously, they were deposited in the Protein Data Bank without accompanying publications. All of them have uracil bound in the effector‐binding site, representing the inactive form of the regulator. We determined the crystal structure of an unliganded mutant RutR protein and provide a structural basis for the use of uracil as sole effector molecule and the exclusion of the very similar thymine from the ligand‐binding pocket.

Keywords

• carAB
• Arginine biosynthesis
• Pyrimidine biosynthesis
• Carbamoylphosphate
• Protein–DNA interactions
• Crystallography