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The Effect of Self-association on the Interaction of theEscherichia coliRegulatory Protein TyrR with DNA

1996; Elsevier BV; Volume: 263; Issue: 5 Linguagem: Inglês

10.1006/jmbi.1996.0607

1089-8638

Michael F. Bailey, Barrie E. Davidson, Allen P. Minton, William H. Sawyer, Geoffrey J. Howlett,

RNA and protein synthesis mechanisms

The interaction of theEscherichia coliregulatory 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) × 106M−1) was determined by a global analysis of sedimentation data, collected at multiple wave- lengths between 230 and 285 nm. The presence of 30 μM ATPγS enhanced the affinity of TyrR for DNA approximately 3.5-fold, (Ko,d=9.9(±0.3) × 106M−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 × 106M−1), and to either one or three sites on the hexamer (Ko,h=2.0(±0.1) × 108M−1and 3.8(±0.1) × 106M−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.