CRITICAL BASE-PAIRS AND AMINO-ACID-RESIDUES FOR PROTEIN-DNA INTERACTION BETWEEN THE TYRR PROTEIN AND TYRP OPERATOR OF ESCHERICHIA-COLI

In Escherichia coli K-12, the repression of tyrP requires the binding of the TyrR protein to the operator in the presence of coeffectors, ty rosine and ATP, This operator contains two 22-bp palindromic sequences which are termed TyrR boxes, Methylation, uracil, and ethylation inte rference experiments were used to identify the important sites in the TyrR boxes that make contacts with the TyrR protein, Methylation inter ference studies demonstrated that guanines at positions +8, -5, and -8 of the strong TyrR box and positions +8, -4, and -8 of the weak box a re close to the TyrR protein, Uracil interference revealed that strong van der Waals contacts are made by the thymines at position -7 and +5 of the top strands of both strong and weak bones and that weaker cont acts are made by the thymines at positions +7 (strong box) and -5 and +7 (weak box) of the bottom strand, In addition, ethylation interferen ce suggested that the phosphate backbone contacts are located at the e nd and central regions of the palindrome, These findings are supported by our results derived from studies of symmetrical mutations of the t yrP strong box, Overall, the results confirm the critical importance o f the invariant (G . C)(C . G)8 base pairs for TyrR recognition and al so indicate that interactions with (T . A)(A . T)7 are of major import ance, In contrast, mutations in other positions result in weaker effec ts on the binding affinity of TyrR protein, indicating that these posi tions play a lesser role in TyrR protein recognition, Alanine scanning of both helices of the putative helix-turn-helix DNA-binding motif of TyrR protein has identified those amino acids whose side chains play an essential role in protein structure and DNA binding.