title: The effect of self-association on the interaction of the Escherichia coli regulatory protein TyrR with DNA.
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.
authors.affiliation: Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: M F
authors.initials: MF
authors.lastname: Bailey
authors.forename: B E
authors.initials: BE
authors.lastname: Davidson
authors.forename: A P
authors.initials: AP
authors.lastname: Minton
authors.forename: W H
authors.initials: WH
authors.lastname: Sawyer
authors.forename: G J
authors.initials: GJ
authors.lastname: Howlett
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D012097:Repressor Proteins; C069256:TyrR protein, E coli; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate