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

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 (K-o,K-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 mu M ATP gamma S enhanced the affinity of TyrR for DNA approximately 3.5-fold, (K-o,K-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 (k(o,d) = 9.9 x 10(6) M(-1)), and to either one or three sites on the hexamer (K-o,K-h = 2.0(+/-0.1) x 10(8) 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. (C) 1996 Academic Press Limited