title: Ligand binding specificity of RutR, a member of the TetR family of transcription regulators in Escherichia coli.
abstract: RutR is a member of the large family of TetR transcriptional regulators in Escherichia coli. It was originally discovered as the regulator of the rutABCDEFG operon encoding a novel pathway for pyrimidine utilization, but its highest affinity target is the control region of the carAB 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 on in vitro DNA binding in absence and presence of potential ligands, and analyse their effect on positive regulation of the carP1 promoter and negative autoregulation in 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.
authors: Research Group of Microbiology, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussel, Belgium ; Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Centro de Investigación Biomédicaen Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, E-46010 Valencia, Spain.
authors: Phu
authors: P
authors: Nguyen Le Minh
authors: Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Centro de Investigación Biomédicaen Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, E-46010 Valencia, Spain.
authors: Sergio
authors: S
authors: de Cima
authors: Research Group of Microbiology, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussel, Belgium.
authors: Indra
authors: I
authors: Bervoets
authors: Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussel, Belgium.
authors: Dominique
authors: D
authors: Maes
authors: Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Centro de Investigación Biomédicaen Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, E-46010 Valencia, Spain.
authors: Vicente
authors: V
authors: Rubio
authors: Research Group of Microbiology, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussel, Belgium.
authors: Daniel
authors: D
authors: Charlier
keywords: Arginine biosynthesis; Carbamoylphosphate; Crystallography; Protein–DNA interactions; Pyrimidine biosynthesis; carAB