Mechanism of upstream promoter element stimulation of transcription at a ribosomal RNA promoter determined by single-molecule imaging

Abstract

DNA elements upstream of transcription promoters play a role in regulating transcription initiation in all organisms. In bacteria, upstream A-T rich sequences called UP elements can stimulate transcription through contact with the α subunit C-terminal domain (αCTD) of core RNA polymerase (RNAP), but the kinetic mechanisms by which they do so remain unclear. We investigated the role of the UP element in stimulating initiation from the strong E. coli 16s rRNA promoter using single-molecule fluorescence microscopy to visualize σ70RNAP holoenzyme binding and the formation nascent RNA by oligonucleotide probe hybridization on individual DNA molecules containing the rrnB P1 promoter. By directly detecting initial binding of σ70RNAP to promoter and monitoring the lifetimes of promoter-polymerase complexes, the experiments reveal the kinetic mechanism of polymerase recruitment to the promoter and the subsequent conformational change that stabilizes binding. The presence of UP stimulated the rate of initial binding of polymerase to promoter by at least six-fold, and this stimulation was fully sufficient to account for the increase in initiation rate by UP. Thus, UP likely functions at this strong promoter simply by acting as a binding target for the rapidly reorienting αCTD domain tethered to the core polymerase. In contrast, there were only minor effects of UP on the measured rates of the conformational change or the dissociation rates of the initial σ70RNAP promoter complexes. These studies define a paradigmatic kinetic mechanism for stimulation of transcription initiation by direct αCTD-DNA interactions. This mechanism can serve as a building block of more complex regulatory architectures in which αCTD promotes transcription through interactions with both DNA and protein activators.