MECHANISMS OF UPSTREAM ACTIVATION OF THE RRND PROMOTER P(1) OF ESCHERICHIA-COLI

The rrn promoter regions of Escherichia coli contain a stretch of DNA, rich in A(n) and T(n) homopolymer sequences, which is located upstrea m of the tandem promoters P1 and P2. We have studied the effects of th e upstream sequence of the rrnD operon on promoter function, using del etion variants for in vitro transcription. The presence of the upstrea m activating sequence (UAS) was found to increase P1 promoter strength without influencing P2. Two modes of P1 activation could be distingui shed: a stimulation of P1 depending on the interaction of the factor o f inversion stimulation (FIS) with the UAS (within the deletion bounda ries of -50 and -112) and second, a factor-independent activation requ iring the proximal part of the UAS (within the boundaries of -50 and - 89). Both modes of activation were previously observed in the case of the rrnB operon and were ascribed to increased constants of RNA polyme rase binding to P1 (Leirmo, S., and Gourse, R. L. (1991) J. Mol. Biol. 220, 555-568; Zacharias, M., Theissen, G., Bradaczek, C., and Wagner, R. (1991) Biochimie 73, 699-712). Our results show, however, that the mechanisms of upstream activation may vary with the reaction conditio ns. In a complex transcription system, originally designed for the use of cell extracts, FIS enhances first-order reactions that convert bin ary (open) complexes to transcribing complexes. Initiated complexes ar e stabilized by FIS. The factor-independent mode of P1 activation invo lves influences of the UAS on complex isomerizations as well as on bin ary complex formation. The results show that low molecular weight comp onents of the complex transcription system change the function of the RNA polymerase at the rrn promoters (not at the reference promoter P(t ac)), so that the conversion of open to transcribing P1-complexes beco mes dependent on the UAS and FIS.