Sliding and intermolecular transfer of the lac repressor: kinetic perturbation of a reaction intermediate by a distant DNA sequence.
The lac repressor associates with its operator at a rate faster than conventional diffusion allows, either because of one-dimensional diffusion of a captured repressor along the DNA (sliding) or because the tetrameric repressor can be rapidly transferred between DNA sites distant from each other in the primary sequence (direct transfer). We report measurements of relative repressor-operator association rates at physiological operator concentrations. We assay for the ability of DNA targets of equal length (approximately 200 base pairs) containing one or six operator segments to compete for repressor; as the sliding range decreases, the hexameric operator fragment should react up to six times faster than the monomeric operator fragment. We find that the advantage enjoyed by the hexameric fragment varies from little to none at low operator concentration (depending on ionic strength) to more than 3-fold at high concentration. We ascribe this behavior to sliding at low operator concentration and to an increasing contribution of bimolecular direct transfer events as concentration increases. The observations require a "semibound" intermediate state of the protein at operator sites. This species can either undergo a relatively slow (tau greater than 1 sec) unimolecular isomerization to the final complex, or the isomerization can occur in a bridged complex with another operator site, accompanied by transfer to the second operator with probability of 0.5. Bridging alters one or more rate constants in the complex.
Ruusala
T
T
Department of Chemistry, Yale University, New Haven, CT 06511.
Crothers
D M
DM
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D004058:Diffusion; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins