RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates.

Abstract

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires sigma factor that participates in both promoter recognition and DNA melting. RNAP lacking sigma (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various sigma factors. Using bubble templates containing the sigmaD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of sigma. This supports a model in which the UP element acts primarily through the alpha subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: sigmaD restricts initiation to sites 8-9 nucleotides downstream of the conserved -10 element. Remarkably, sigmaA also has a dramatic effect on start-site selection even though the sigmaA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the sigmaA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved -10 hexamer recognized by sigmaA. Thus, sigmaA appears to recognize the -10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, sigma also localizes the transcription start site.