Cooperative binding to DNA of catabolite activator protein of Escherichia coli

Saxe, Stephen A.; Revzin, Arnold

doi:10.1021/bi00569a003

Cited by 81 publications

(58 citation statements)

References 39 publications

(36 reference statements)

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“…CAP has been shown to interact cooperatively with RNA polymerase at the lac and gal promoters (18,23,(28)(29)(30) and with the lac repressor at the lac promoter (15). In addition to these heterologous interactions, CAP interacts cooperatively with itself when it binds specifically to the gal promoter (28) in the presence of cAMP and when it binds nonspecifically to DNA in the absence of cAMP (13,25,32 10 mM MgCI2, and 100 mM KCl. The -2-fold increase in K11K2 accompanying the addition of 50 mM potassium glutamate to the reaction buffer indicates that K1 and K2 have different salt dependencies.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…An 8.40C increase was observed for the saturation of non-specific sites on the 62 bp DNA. The larger increase for the 62 bp DNA may be due to the more cooperative binding of CAP without cAMP (18), and/or the larger relative influence of the fragment ends.…”

Section: Discussionmentioning

confidence: 99%

“…Base pair opening will also increase and will deplete duplex DNA sltes. Since CAP binds weakly to single stranded DNAs (18), the amount of free CAP, susceptable to rapid denaturation, will rise. For a situation where Tm > TD, CAP may become inactive as soon as base pair opening becomes significant.…”

Section: Discussionmentioning

confidence: 99%

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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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“…Concentrations were determined by absorption spectroscopy at 278 nm using E = 1.99 x lo4 M-' [30] for CRP monomers.…”

Section: Protein Solutionsmentioning

confidence: 99%

Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli

Donoso

Turner

King

1987

European Journal of Biochemistry

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cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6-NHz group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of CAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent. The 2-NHz group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent.CAMP-receptor (CRP), also referred to as catabolite gene activator protein (CAP), regulates the transcription of catabolite-sensitive genes in Escherichia coli and other prokaryotes [l -41. To act, CRP requires cAMP as an allosteric activator [5]. The CRP-CAMP complex binds to specific sites on DNA at or near promoters, either stimulating the initiation of RNA synthesis [6 -81 to produce positive regulation, or blocking the action of RNA polymerase producing negative regulation, as in the case of its own structural gene [9].CRP is a dimeric protein composed of two chemically identical subunits each of relative molecular mass 23619 as deduced from the nucleotide sequence [lo, 111. McKay et al. [12, 131 have determined the X-ray crystal structure of the CRP-CAMP complex at a resolution of 0.29 nm and have shown that each subunit consists of two structural domains. The larger amino-terminal domain contains the cAMP binding site while the smaller carboxy-terminal domain forms part of the DNA binding site.There is evidence that cAMP binding to CRP induces a conformational change in the protein. For example, the complex is easily cleaved by trypsin, chymotrypsin, Staphylococcus aureus V8 protease and subtilisin, whereas unliganded CRP is resistant to proteolysis [14, 151. In the presence of 5',5'-dithiobis(2-nitrobenzoic acid) the complex forms an intersubunit cysteine-cysteine subunit, whereas CRP alone does not [16]. cAMP binding to AENS-labelled CRP induces changes in fluorescence intensity and a blue shift in the emission maximum, and also alters the relaxation time of the labelled protein in temperature-jump experiments [17, 181. Small-angle X-ray scattering experiments show a decrease of 0.4 nm in the radius of gyration of CRP when cAMP binds [19]. Proton magnetic resonance studies [20] have also shown

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Cooperative binding to DNA of catabolite activator protein of Escherichia coli

Cited by 81 publications

References 39 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

Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli

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