Sequence-specific DNA displaces 6-p-toluidino-2-naphthalenesulfonate bound to a hydrophobic site on the DNA-binding domain of Drosophila c-myb
1994
Madan, A. | Hosur, R.V. | Padhy, L.C.
The N-terminal DNA-binding domain of c-myb oncoprotein binds to DNA in a sequence-specific manner. The domain, consisting of three imperfect tandem repeats, has tryptophan residues at very regular intervals and this is believed to be of some significance in the DNA-binding activity of the protein. We have found that the hydrophobic-site-specific probe 6-p-toluidino-2-naphthalenesulfonate (TNS) binds to the bacterially expressed DNA-binding domain of Drosophila c-myb protein (R123). TNS has a single binding site on this protein with an apparent dissociation constant in the range of (5-8) X 10(-7) M. When the TNS-protein complex was treated with an oligomeric DNA duplex having a cognate myb-binding site, the TNS was displaced from the complex. Nonspecific DNA duplex oligomers were ineffective, indicating that TNS displacement was a sequence-specific process. We examined further some features of the TNS-binding site on the protein, taking advantage of the fluorescence properties of the protein and the bound TNS. Our data indicate that the TNS binding occurs in a peripheral site on the protein in a manner that allows the bound TNS to be solvent accessible. Furthermore, there are indications that tyrosine(s) and tryptophans of the protein mediate resonance energy transfer to the bound TNS. From fluorescence-quenching data of the protein and protein-TNS complex, we could assess that both solvent-accessible and internal tryptophans are in the vicinity of the bound TNS. Our data are consistent with the proposal that the bound TNS occupies a part of the DNA-binding site of the protein and is made up of contributions from at least two of the three typical repeating units of Dm-myb protein.
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