INTERACTION BETWEEN THE ESCHERICHIA-COLI REGULATORY PROTEIN TYRR AND DNA - A FLUORESCENCE FOOTPRINTING STUDY

The Escherichia coil regulatory protein TyrR controls the expression o f eight transcription units that encode proteins involved in the biosy nthesis 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 in duces self-association of TyrR from a dimer to a hexamer. This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide cont aining a centrally located binding site for TyrR (TyrR box). Replaceme nt 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 oligonucleoti de was followed using steady-state and time-resolved fluorescence meth ods. Binding of the TyrR dimer caused significant changes in the fluor escent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protei n. Except for the position 15 conjugate, binding of the TyrR dimer cau sed little change in fluorescence intensity. Therefore, fluorescence a nisotropy was used to follow the binding equilibium. The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, sugge sting that the fluorophore was in close contact with the protein. For all conjugates, the addition of tyrosine at the end of the titration w ith TyrR increased the anisotropy markedly, suggesting that the hexame ric form of TyrR could bind the oligonucleotide, Two rotational correl ation 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 oligonucleot ide (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 prese nted that the binding of the TyrR hexamer, but not the TyrR dimer, aff ects regions that flank the binding sequence. The results support the hypothesis that the binding of the TyrR hexamer is responsible for int eraction between tandem TyrR boxes in the tyrR regulon.