title: Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.
abstract: Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.
authors.affiliation: Department of Medical Virology, Uppsala University Biomedical Centre, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Bondeson
authors.forename: A
authors.initials: A
authors.lastname: Frostell-Karlsson
authors.forename: L
authors.initials: L
authors.lastname: Fägerstam
authors.forename: G
authors.initials: G
authors.lastname: Magnusson
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D004269:DNA, Bacterial / Q000378:metabolism*; D007700:Kinetics; D007763:Lac Operon; D008433:Mathematics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins