title: Probing co-operative DNA-binding in vivo. The lac O1:O3 interaction.
abstract: The lac primary (O1) and weak upstream pseudo (O3) operators contained on a plasmid were footprinted in vivo in order to determine whether they act co-operatively in binding lac repressor in the cell. The occupancy at O3 by lac repressor was substantially reduced upon deletion of the lac primary operator, demonstrating co-operativity at a distance. Plots of operator occupancy versus active repressor concentration were obtained for each operator by treating the cells with different amounts of the lac inducer isopropyl-beta-D-thiogalactoside and probing lac repressor binding. This analysis can be used to obtain relative binding constants in vivo and demonstrates that O3 binds repressor only 10.3-fold less tightly than O1 in their co-operative interaction. The removal of DNA torsional tension in vivo by the use of coumermycin leads to the same loss of binding at O3 as does deleting O1. These in-vivo results are analogous to the in-vitro situation, where O3 binds repressor strongly in a DNA repression loop only on supercoiled templates.
mesh_terms: D000494:Allosteric Regulation; D001345:Autoradiography; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000493:pharmacokinetics; D007763:Lac Operon; D012097:Repressor Proteins / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside