Cation binding linked to a sequence-specific CAP–DNA interaction

Abstract

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.

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

“…The complex consists of a single molecule of CAP bound predominantly to the highest affinity CAP site in the lac promoter (CAP site 1 49,66 ). Electrophoresis was carried out with a 10% w/v polyacrylamide (75:1 acrylamide:bisacrylamide) gel, cast and run in the Tris-acetate EDTA buffer described in the protocol shown in Table 5.…”

“…The assay is not limited by the salt concentration of the sample and it can accommodate very large nucleic acids (e.g., the phage λ genome (48,502 bp 46 ) 47 . In contrast, the electrophoretic process limits the salt concentrations of EMSA samples to ≤300 mM (1:1 salt) and to DNAs of 5000 bp 18,48,49 (note that these limits are approximate and may vary with sample and gel compositions). The presence of more than one binding protein complicates filter binding analysis, since retention of labeled nucleic acid is detected, not the identity of bound proteins or the proportion of binding activity attributable to each.…”

“…The protein is purified E. coli cyclic AMP receptor protein (CAP) 49,67 and the nucleic acid is a 214 bp restriction fragment from the E. coli lac promoter-operator region 48 . The binding buffer consists of 10 mM Tris (pH 7.5 at 20°C), 100 mM KCl, 1 mM EDTA, 0.1 mM DTT, 20 μM cyclic AMP, 5% v/v glycerol, 0.010 mg/mL BSA and the electrophoresis was carried out in a 10% w/v polyacrylamide (75:1 acrylamide:bisacrylamide) gel, cast in 40 mM Tris-acetate, 2.5 mM EDTA (pH 7.8 at 20°C) and run in 40 mM Tris-acetate, 2.5 mM EDTA (pH 7.8 at 20°C), supplemented with 20μM cyclic AMP.…”

Assembly of Functional Ribonucleoprotein Complexes by AU-rich Element RNA-binding Protein 1 (AUF1) Requires Base-dependent and -independent RNA Contacts

“…tions His 6 -p37 AUF1 binding was also impaired, suggesting that specific macromolecular interactions with ions in solution could also be required to stabilize p37 AUF1 RNP complexes or to maintain the protein in an active conformation as has been postulated for binding of the E. coli CAP and AraC proteins to cognate DNA targets (46,60). To incorporate broader roles for solvated ions in p37 AUF1 RNP assembly, logK obs versus log[KCl] plots were analyzed using a more inclusive counterion binding model described by Equation 14 (Fig.…”

“…is related to a parameter termed , which is sequence-, pH-, and counterion-dependent and proportional to the structural charge density (42,43 (46,47). This model includes changes in the numbers of protein-associated cations (⌬m) and anions (⌬n) resulting from protein binding to the nucleic acid target in addition to the change in cations associated with the nucleic acid (⌬q).…”