Interaction between the Escherichia coli Regulatory Protein TyrR and DNA: A Fluorescence Footprinting Study

Abstract

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 is a homodimer of 57 600 subunit molecular weight and has a binding site for ATP and weak ATPase activity. In the presence of ATP, TyrR binds tyrosine, which induces self-association of TyrR from a dimer to a hexamer. This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide containing a centrally located binding site for TyrR (TyrR box). Replacement of a thymidine residue with an aminouridine residue at positions 7, 9, 13, 15, 19, 22, and 26 from one end of the 42mer enables labeling with fluorescein and successive placement of the label along the major groove of the DNA. The fluorescence footprinting of the oligonucleotide was followed using steady-state and time-resolved fluorescence methods. Binding of the TyrR dimer caused significant changes in the fluorescent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protein. Except for the position 15 conjugate, binding of the TyrR dimer caused little change in fluorescence intensity. Therefore, fluorescence anisotropy was used to follow the binding equilibrium. The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, suggesting that the fluorophore was in close contact with the protein. For all conjugates, the addition of tyrosine at the end of the titration with TyrR increased the anisotropy markedly, suggesting that the hexameric form of TyrR could bind the oligonucleotide. Two rotational correlation times were found for the labeled conjugates: one reflecting the motion of the probe at its point of attachment to the DNA (220-290 ps), the other reflecting the global tumbling of the labeled oligonucleotide (14-21 ns). On binding TyrR, changes in the correlation times and their associated amplitudes and changes in the range of angular motion of the probe depended on the position of the label. Evidence is presented that the binding of the TyrR hexamer, but not the TyrR dimer, affects regions that flank the binding sequence. The results support the hypothesis that the binding of the TyrR hexamer is responsible for interaction between tandem TyrR boxes in the tyrR regulon.

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

“…In our study, the values of naked ODNs are relatively low as compared with those found by other groups (27)(28)(29). The origin of this discrepancy is not clear but our data show that the steady-state anisotropy is not proportional to the ODN length.…”

“…Other examples of protein-DNA complexes described in the literature display similar behavior. For TBP (27 kDa) and the TyrR homodimer (57.6 kDa), the values of the final protein-DNA complexes (8.5 and 28 ns, respectively) are similar to those predicted for the free proteins (27,28).…”

Thermodynamic Dissection of the Polymerizing and Editing Modes of a DNA Polymerase

“…In a previous study, the association of TyrR protein with a 42 bp DNA duplex containing a fluorescein label adjacent to the protein-binding site also resulted in a significant fluorescence enhancement and an increase of anisotropy. 23 In that system, the fluorescence changes were attributed to contact between the protein surface and the fluorophore, and it is likely that the fluorescence behavior observed here is also the result of a direct interaction between the probe and the protein. This is somewhat surprising, because the fluorescein probe is attached to the 5 0 end of the primer strand, some distance upstream of the junction between the double and single-stranded regions of the primer/template where KF is expected to bind.…”

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

“…One such study with template/primer oligonucleotides, where a fluorescent dansyl probe was appended at various nucleotide positions from the 3 -end of the primer, indicated that the Klenow polymerase remains in contact with the template/primer up to six nucleotides from the 3 -end (238). Similarly, contacts between a 42-mer oligonucleotide and the E. coli regulatory protein TyrR were also identified through the use of such nucleoside analogs (239).…”