Cation binding linked to a sequence-specific CAP-DNA interaction.
The equilibrium association constant observed for many DNA-protein interactions in vitro (K(obs)) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that release of cations from the DNA is the dominant involvement of ions in the binding reaction. With this assumption, the graph of logK(obs) versus log[MX] is predicted to have a constant slope proportional to the number of ions released from the DNA upon protein binding. However, experimental data often deviate from log-linearity at low salt concentrations. Here we show that for the sequence-specific interaction of CAP with its primary site in the lactose promoter, ionic stoichiometries depend strongly on cation identity and weakly on anion identity. This outcome is consistent with a simple linkage model in which cation binding by the protein accompanies its association with DNA. The order of ion affinities deduced from analysis of DNA binding is the same as that inferred from urea-denaturation experiments performed in the absence of DNA, suggesting that ion binding to free CAP contributes significantly to the ionic stoichiometry of DNA binding. In living cells, the coupling of ion-uptake and DNA binding mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.
Stickle
Douglas F
DF
Department of Pathology and Microbiology, 986495 Nebraska Medical Center, Omaha, NE 69198-6495, USA.
Fried
Michael G
MG
D000595:Amino Acid Sequence; D000838:Anions / Q000737:chemistry; D001483:Base Sequence; D002412:Cations / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D011953:Receptors, Cyclic AMP / Q000737:chemistry*; D012492:Salts / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D000838:Anions; D002412:Cations; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011953:Receptors, Cyclic AMP; D012492:Salts; C047947:cyclic AMP receptor protein, E coli; D014508:Urea; D004247:DNA