RNA-POLYMERASE SIGMA-FACTOR DETERMINES START-SITE SELECTION BUT IS NOT REQUIRED FOR UPSTREAM PROMOTER ELEMENT ACTIVATION ON HETERODUPLEX (BUBBLE) TEMPLATES

Sequence-selective transcription by bacterial RNA polymerase (RNAP) re quires a 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 u sed DNA templates containing short regions of heteroduplex (bubbles) t o compare initiation in the presence and absence of various sigma fact ors, Using bubble templates containing the sigma(D)-dependent flagelli n promoter, with or without its associated upstream promoter (UP) elem ent, we demonstrate that UP element stimulation occurs efficiently eve n in the absence of sigma. This supports a model in which the UP eleme nt acts primarily through the alpha subunit of core enzyme to increase the initial association of RNAP with the promoter, Core and holoenzym e do differ substantially in the template positions chosen for initiat ion: sigma(D) restricts initiation to sites 8-9 nucleotides downstream of the conserved -10 element, Remarkably, sigma(A) also has a dramati c effect on start-site selection even though the sigma(A) holoenzyme i s inactive on the corresponding homoduplexes, The start sites chosen b y the sigma(A) holoenzyme are located 8 nucleotides downstream of sequ ences on the nontemplate strand that resemble the conserved -10 hexame r recognized by sigma(A), Thus, sigma(A) 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, s igma also localizes the transcription start site.