Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli

In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine an d thymine bases and phosphates within the TyrR box sequences that are conta cted by the TyrR protein (J. S. Hwang, J, Yang, and A, J, Pittard, J. Bacte riol. 179:1051-1058, 1997). In this study, we have used missing contact pro bing to test the involvement of all of the bases within the tyrP operator i n the binding of TyrR. Our results indicate that nearly all the bases withi n the palindromic arms of the strong and weak boxes are important for the b inding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, H A494 and TA495, were purified, and their binding affinities for the tyrP op erator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison w ith wild Type TyrR, TA495 had only a small reduction in DNA binding. Missin g contact probing was performed by using the purified TA195 protein, and th e results suggest that T495 makes specific contacts with adenine and thymin e bases at the +/-5 positions in the TyrR boxes.