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

Jeffrey T. Owens, Albert J. Chmura, Katsuhiko Murakami, Nobuyuki Fujita, Akira Ishihama, Claude F. Meares

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)7670-7675
Number of pages6
JournalBiochemistry
Volume37
Issue number21
DOIs
StatePublished - May 26 1998

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ASJC Scopus subject areas

  • Biochemistry

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