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 and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator 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 with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes.
Hwang
J S
JS
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia.
Yang
J
J
Pittard
A J
AJ
D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D009154:Mutation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism*
D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011687:Purines; D011743:Pyrimidines; D012097:Repressor Proteins; C069256:TyrR protein, E coli; C057333:tyrP protein, E coli