Factor-independent activation of Escherichia coli rRNA transcription : II. Characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase

Abstract

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E σ 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E σ 70 complexes formed in the presence of the initiating nucleotides (RP init) differ from typical open complexes (RP o) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RP init complex closely resembles open complexes formed at other E σ 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position −18 in both RP o and RP init indicates the possible presence of a subtle difference in the −10, −35 spacer structure compared to some other E. coli promoters. We show that the E σ 70- rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the −50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E σ 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E σ 70.