Distances between DNA and ATP Binding Sites in the TyrR−DNA Complex

• Published in: Biochemistry (Easton) 2000-05, Vol.39 (19), p.5653-5661
• Main Authors: Sawyer, William H, Chan, Robert Y. S, Eccleston, John F, Davidson, Barrie E, Samat, Saiffudin A, Yan, Yuling
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Binding Sites; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; Energy Transfer; Escherichia coli; Escherichia coli - chemistry; Escherichia coli Proteins; Fluorescence Polarization; Macromolecular Substances; Models, Molecular; Oligonucleotides - chemistry; Oligonucleotides - metabolism; Repressor Proteins - chemistry; Repressor Proteins - metabolism; Rhodamines - chemistry; Rhodamines - metabolism; Spectrometry, Fluorescence; TyrR protein
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0000723

The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It binds to DNA as a homodimer with a subunit molecular mass of 57 640 Da, each of which has a single site for the binding of ATP within a central structural domain. This paper reports distances between four sites on the DNA and the ATP binding site as determined by fluorescence resonance energy transfer. The DNA was a 30mer containing a centrally located binding site for TyrR. Replacement of a thymidine residue with an aminouridine residue at positions −9, −7, −3, and 2 of the palindromic oligonucleotide sequence enabled the placement of a single fluorescein group along the major groove of the DNA. The energy transfer acceptor was ATP labeled with a rhodamine group through positions 2‘ and 3‘ of the ribose, positions that are known to cause minimal interference with the binding of ATP to protein. The dissociation constant for the binding of rhodamine-ATP to TyrR was 300 nM as determined by steady-state fluorescence anisotropy titrations. The energy transfer efficiencies were determined by measuring the level of quenching of donor fluorescence on binding rhodamine-ATP to the TyrR−DNA complex. The experimental transfer efficiencies were compared to theoretical values calculated for a model of the DNA−TyrR complex in which the position of the ATP binding site was allowed to vary over the surface of the monomer unit. Theory was written to account for the transfer from one donor to two acceptors, one on each monomer unit of the TyrR dimer. The results indicate that the ATP binding site is about 40−45 Å from the nearest point on the DNA and distant from the DNA helix−turn−helix binding domain. The effects of ATP binding of (i) increasing the TyrR binding affinity by a factor of 4−5 and (ii) permitting the binding of the tyrosine corepressor must therefore occur because of a significant allosteric change in the conformation of the protein.