Mapping the promoter DNA sites proximal to conserved regions of σ70 in an Escherichia coli RNA polymerase-lacUV5 open promoter complex

Base-specific interactions between promoter DNA and Escherichia coli RNA polymerase are regulated by a sigma (a) protein during transcription initiation. To map spatial relations between evolutionarily conserved regions of the primary sigma (sigma(70)) and each DNA strand along the lacUV5 promoter in the transcriptionally active "open" complex, we have used a cysteine-tethered cutting reagent to cleave DNA strands. The chemical nuclease FeBABE [iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate] was conjugated to single-cysteine mutants of sigma(70) at sites 132C, 376C, 396C, 422C, 496C, 517C, or 581C. After formation of open promoter complexes between lacUV5 DNA and RNA polymerase holoenzymes carrying conjugated sigma(70) subunits, we observed promoter DNA cleavage spanning at least 60 bases, between positions -48 and +12. The results show that sigma(70) region 2.1, otherwise implicated in core enzyme binding, is proximal to the nontemplate strand of lacUV5 DNA between the -10 promoter element and positions as far downstream of the transcription start site as +12. Conserved region 3.2 of sigma 70 is proximal to the template strand near the +1 transcription start site, and region 3.1 is positioned between the lacUV5-10 and -35 promoter elements. We propose a model for the orientation of sigma(70) and DNA in the open complex.