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 (σ) protein during transcription initiation. To map spatial relations between evolutionarily conserved regions of the primary sigma (σ⁷⁰) 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)-l-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate] was conjugated to single-cysteine mutants of σ⁷⁰ 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 σ⁷⁰ subunits, we observed promoter DNA cleavage spanning at least 60 bases, between positions -48 and + 12. The results show that σ⁷⁰ 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 σ⁷⁰ 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 σ⁷⁰ and DNA in the open complex.