Factor-independent activation of Escherichia coli rRNA transcription. I. Kinetic analysis of the roles of the upstream activator region and supercoiling on transcription of the rrnB P1 promoter in vitro.

The region from position -154 to position -50 upstream from the start site of transcription of the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), is required for maximal promoter activity in vivo. Maximal activation (20 to 30-fold) requires the binding of Fis protein in vitro and in vivo. However, two- to fourfold activation remains in vivo even in the absence of Fis. Here, we demonstrate that the presence of the UAR increases the rate of formation of E sigma 70-promoter complexes in vitro in the absence of added protein factors (factor-independent activation). The UAR increases the rate of the RNA polymerase concentration-dependent step in the association pathway to a stable complex formed in the presence of the initiating nucleotides ATP and CTP (RPinit). The rate of dissociation from RPinit is not affected. In addition, a supercoiled template of native superhelical density increases both the association rate for the formation of RPinit and the lifetime of complexes formed in the absence of nucleotides (RPo or open complex), but does not affect factor-independent activation. The data are consistent with a model whereby the UAR affects only the initial recognition event (closed complex formation) without affecting either the rate or extent of isomerization to the locally denatured open complex. In the accompanying paper, a variety of chemical and enzymatic probes are used to characterize RPinit and RPo both with and without the UAR.