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J Mol Biol. 1996 Nov 15;263(5):671-84.

The effect of self-association on the interaction of the Escherichia coli regulatory protein TyrR with DNA.

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  • 1Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.

Abstract

The interaction of the Escherichia coli regulatory protein TyrR, with a 42 bp oligonucleotide (42A/42B) containing a centrally located recognition sequence (TyrR box), was examined by analytical ultracentrifugation. The stoichiometry of the binding of oligonucleotide to dimeric TyrR was determined by equilibrium centrifugation of a mixture of fluorescein-5-isothiocyanate-labelled 42A/42B (F-42A/42B) in the presence of an eightfold molar excess of TyrR. The molecular mass (M) of the labelled oligonucleotide was estimated as 148,000, indicating a 1:1 complex composed of oligonucleotide (M = 27,000) and TyrR dimer (M = 113,000). The association constant (Ko,d = 2.8(+/- 0.1) x 10(6) M-1) was determined by a global analysis of sedimentation data, collected at multiple wavelengths between 230 and 285 nm. The presence of 30 microM ATP gamma S enhanced the affinity of TyrR for DNA approximately 3.5-fold, (Ko,d = 9.9(+/- 0.3) x 10(6) M-1). The effect of dimer to hexamer self-association of TyrR on the binding of 42A/42B was also examined. Multiple wavelength sedimentation data fitted a model in which the oligonucleotide could bind to one site on the dimer (Ko,d = 9.9 x 10(6) M-1), and to either one or three sites on the hexamer (Ko,h) = 2.0(+/- 0.1) x 10(6) M-1 and 3.8(+/- 0.1) x 10(6) M-1, respectively). Competitive sedimentation equilibrium and fluorescence anisotropy titrations were performed under stoichiometric conditions to resolve the number of oligonucleotide binding sites per hexamer. In these experiments, 42A/42B was used as a competitor to displace F-42A/42B from the hexamer, which was found to bind the 42mer with a 1:1 stoichiometry. Our data support a model in which ATP increases the affinity of TyrR for the DNA recognition sequence, and tyrosine induced self-association of TyrR generates a hexameric species with a single binding site for the 42A/42B oligonucleotide.

PMID:
8947567
DOI:
10.1006/jmbi.1996.0607
[PubMed - indexed for MEDLINE]
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