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

9 Páginas, 6 figuras, 1 tabla. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fob.2015.01.002.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.The authors acknowledge financial support from: Research Foundation Flanders (FWO-Vlaanderen) [G.0429.06 to D.C., a postdoctoral fellowship to P.N.L.M., and a stay abroad to P.N.L.M.]; Research Council of the Vrije Universiteit Brussel (OZR-VUB); Vlaamse Gemeenschapscommissie; Spanisch Government [BFU2011-30407]; Valencian Government [Prometeo II/2014/ 029]; Instituto de Salud Carlos III-CIBERER [contract to S.D.C.].Peer reviewe