Cooperative protein-DNA interactions: effects of KCl on lambda cI binding to OR.
The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of [KCl]. The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.
Koblan
K S
KS
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Ackers
G K
GK
D010582:Bacteriophage lambda / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009876:Operon; D009994:Osmolar Concentration; D010957:Plasmids; D011189:Potassium Chloride / Q000494:pharmacology; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D011189:Potassium Chloride; D003850:Deoxyribonuclease I