title: Optimization of the palindromic order of the TtgR operator enhances binding cooperativity.
abstract: TtgR is the specific transcriptional repressor of the TtgABC efflux pump. TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype of Pseudomonas putida DOT-T1E. TtgR binds to a pseudo-palindromic site that overlaps the ttgR/ttgA promoters. Dimethylsulfate footprint assays reveal a close interaction between TtgR and the central region of this operator. The results of analytical ultracentrifugation demonstrate that TtgR forms stable dimers in solution, and that two dimers bind to the operator. Microcalorimetric analysis of the binding of the two TtgR dimers to the cognate operator showed biphasic behavior, and an interaction model was developed for the cooperative binding of two TtgR dimers to their target operators. The binding of the two TtgR dimers to the operator was characterized by a Hill coefficient of 1.63+/-0.13 (k(D)=18.2(+/-6.3) microM, k(D)(')=0.91(+/-0.49) microM), indicating positive cooperativity. These data are in close agreement with the results of sedimentation equilibrium studies of TtgR-DNA complexes. A series of oligonucleotides were generated in which the imperfect palindrome of the TtgR operator was empirically optimized. Optimization of the palindrome did not significantly alter the binding of the initial TtgR dimer to the operator, but increased the cooperativity of binding and consequently the overall affinity. The minimal fragment for TtgR binding was a 30-mer DNA duplex, and analysis of its sequence revealed two partially overlapping inverted repeats co-existing within the large pseudo-palindrome operator. Based on the architecture of the operator, the thermodynamics of the process, and the TtgR-operator interactions we propose a model for the binding of TtgR to its target sequence.
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado de Correos 419, 18008 Granada, Spain.
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
authors.forename: Wilson
authors.initials: W
authors.lastname: Terán
authors.forename: Obdulio López
authors.initials: OL
authors.lastname: Mayorga
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.forename: Mercedes
authors.initials: M
authors.lastname: Jiménez
authors.forename: Craig
authors.initials: C
authors.lastname: Daniels
authors.forename: Antonio-Jesús
authors.initials: AJ
authors.lastname: Molina-Henares
authors.forename: Manuel
authors.initials: M
authors.lastname: Martínez-Bueno
authors.forename: María-Trinidad
authors.initials: MT
authors.lastname: Gallegos
authors.forename: Juan-Luis
authors.initials: JL
authors.lastname: Ramos
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D018983:DNA Footprinting; D019281:Dimerization; D024901:Drug Resistance, Multiple, Bacterial / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D009875:Operator Regions, Genetic / Q000502:physiology*; D016958:Pseudomonas putida / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C510127:TtgR protein, Pseudomonas putida