On the different binding affinities of CRP at the<i>lac, gal</i>and<i>mal</i>T promoter regions

Kolb, Annie; Spassky, Annick; Chapon, C; Blazy, B.; Buc, Henri

doi:10.1093/nar/11.22.7833

Cited by 166 publications

(98 citation statements)

References 32 publications

(25 reference statements)

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“…Using this method, we find that K1/(K2 ()12) iS 16 within experimental error, the same as that found for K11(K2 W12) when the two sites were present on the same DNA fragment and is consistent with the notion that at low salt concentrations, the cooperativity term W12 1. Parallel experiments performed in the presence of 50 mM potassium glutamate gave a value of K1IK2 = 40.6 (result not shown).…”

Section: Resultssupporting

confidence: 84%

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

Hudson

Fried

1991

J Bacteriol

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The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.

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Section: Resultssupporting

confidence: 84%

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

Hudson

Fried

1991

J Bacteriol

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“…In addition to the primary CAP site, the 144 bp DNA has a second site with a binding constant about 40 fold weaker than the primary site (26). The results of this study were similar to Fig.…”

Section: Introductionsupporting

confidence: 87%

The catabolite activator protein stabilizes its binding site in theE. colilactose promoter

1985

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The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 C.

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“…K3 [3] 102 M-1 determined by direct measurements of cAMP binding (1). However, the significant amount of error in estimating K2 in this study renders (3,4,(15)(16)(17)(18). Fractional activity has also been observed in this laboratory (=20o) when stoichiometric titrations were performed using the gel-shift assay with a 203-bp fragment of lac promoter.…”

Section: Resultsmentioning

confidence: 61%

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

Heyduk

Lee

1990

Proc. Natl. Acad. Sci. U.S.A.

221

195

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A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP4cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.Quantitative structural studies on cAMP receptor protein (CRP) in conjunction with ligand-binding studies have shown clearly that CRP from Escherichia coli exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes (1). The binding properties of these two complexes to the lac promoter were investigated by gelretardation technique, and the results showed that the formation of protein-DNA complex is a complicated function of cAMP concentration. At cAMP concentrations that favor the formation of CRP-cAMP, binding of the protein to DNA is favored. At high concentration of cAMP, which favors the formation of CRP-(cAMP)2, a decrease in protein-DNA complex was seen. These results strongly suggest that the CRP-cAMP and CRP-(cAMP)2 complexes have different affinities for the lac promoter (1). These conclusions are not consistent with the report of Takahashi et al. (2). These authors concluded that the CRP-cAMP complex exhibits essentially the same affinity for the lac promoter as that ofthe CRP-(cAMP)2 complex.The differences between the results of these two studies may be attributed to the differences in experimental conditions, as necessitated by the techniques chosen to monitor protein-DNA interaction. The gel-retardation technique (3,4) dictates that the experiments be conducted at low-salt concentration, and because protein-DNA interactions are highly salt dependent (5), possibly the results of DNAbinding study are not applicable to that of the structure and ligand-binding studies, which are conducted at higher salt concentration (1). To acquire detailed valid thermodynamic data to define the linkages among the interactions of cAMP, CRP, and DNA, we looked for a simple and reliable approach that allows collection of a large amount of accurate data under well...

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On the different binding affinities of CRP at thelac, galandmalT promoter regions

Cited by 166 publications

References 32 publications

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro

The catabolite activator protein stabilizes its binding site in theE. colilactose promoter

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

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