DEMONSTRATION THAT THE TYRR PROTEIN AND RNA-POLYMERASE COMPLEX FORMEDAT THE DIVERGENT P3 PROMOTER INHIBITS BINDING OF RNA-POLYMERASE TO THE MAJOR PROMOTER, P1, OF THE AROP GENE OF ESCHERICHIA-COLI

In previous studies, we have identified three promoters (P1, P2, and P 3) in the regulatory region of the Escherichia coli aroP gene (P. Wang , J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 i s a divergent promoter which overlaps with P1. The repression of trans cription from the major promoter, P1, has been postulated to involve t he activation of the divergent promoter, P3, by the TyrR protein (P. W ang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213-421 8, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence an d absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carryin g the aroP regulatory region, (ii) RNA polymerase has a higher affinit y for P1 than for either P2 or P3 and binds to P1 in the absence of Ty rR protein, (iii) in the presence of TyrR protein and its cofactor, ph enylalanine or tyrosine, RNA polymerase preferentially binds to P3, an d (iv) RNA polymerase does not respond to the activation-defective mut ant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.