Mechanisms of upstream activation of the rrnD promoter P1 of Escherichia coli.

The rrn promoter regions of Escherichia coli contain a stretch of DNA, rich in An and Tn homopolymer sequences, which is located upstream of the tandem promoters P1 and P2. We have studied the effects of the upstream sequence of the rrnD operon on promoter function, using deletion variants for in vitro transcription. The presence of the upstream activating sequence (UAS) was found to increase P1 promoter strength without influencing P2. Two modes of P1 activation could be distinguished: a stimulation of P1 depending on the interaction of the factor of inversion stimulation (FIS) with the UAS (within the deletion boundaries of -50 and -112) and second, a factor-independent activation requiring 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 polymerase 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 conditions. In a complex transcription system, originally designed for the use of cell extracts, FIS enhances first-order reactions that convert binary (open) complexes to transcribing complexes. Initiated complexes are stabilized by FIS. The factor-independent mode of P1 activation involves influences of the UAS on complex isomerizations as well as on binary complex formation. The results show that low molecular weight components of the complex transcription system change the function of the RNA polymerase at the rrn promoters (not at the reference promoter Ptac), so that the conversion of open to transcribing P1-complexes becomes dependent on the UAS and FIS.