Modulation of the stability of a Lac repressor mediated looped complex by temperature and ions: allosteric regulation by chloride.
The lactose repressor of Escherichia coli (LacI) associates to a bidentate tetramer in solution and can simultaneously bind two operators to form a protein-mediated "looped complex". Studies have been conducted of the binding of LacI to two operators separated by approximately 11 helical turns of DNA. Quantitative DNase I footprint titration analysis of the stability of the LacI-mediated looped complex reveals that the Gibbs free energy of cyclization (delta Gzeroj) of the looped complex of 11.7 +/- 0.4 kcal/mol is invariant with temperature. van't Hoff analysis reveals a large and positive enthalpy of cyclization (delta H degrees = 12.3 +/- 2.4 kcal/mol) and an entropy that is small and positive (delta S degrees = 2.2 cal/deg). Quantitative DNase I footprint titration and kinetic dissociation studies were also conducted as a function of counter-ion type and concentration. Increasing concentrations of KCl or potassium glutamate destabilize the looped complex, a result completely accounted for by increases in the intrinsic DNA-binding free energies. While the value of delta Gzeroj is invariant with ion concentration, chloride is a positive regulator. The value of delta Gzeroj decreases by 1.5 kcal/mol upon substitution of chloride for glutamate. Measurements of delta Gzeroj conducted as a function of chloride concentration at constant ionic strength reveal that approximately one chloride ion per tetramer is bound upon looped complex formation. These results demonstrate specific allosteric regulation of the formation of the LacI-mediated looped complex by a mechanism distinct from the regulation of the constituent protein--DNA interactions.
Brenowitz
M
M
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Jamison
E
E
D000494:Allosteric Regulation; D001482:Base Composition; D001483:Base Sequence; D002151:Calorimetry; D002712:Chlorides / Q000378:metabolism* / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions; D013379:Substrate Specificity
D002712:Chlorides; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D012996:Solutions; D003850:Deoxyribonuclease I