Abstract
Initiation of RNA synthesis by bacterial RNA polymerase (RNAP) requires melting of promoter DNA, which is nucleated by the σ subunit during formation of the “open” promoter complex (RPo). The antibiotic lipiarmycin (Lpm) inhibits promoter melting by blocking access of the template DNA strand to the RNAP active-site cleft. Here we show that Escherichia coli RNAP holoenzymes containing either housekeeping σ70, with a deletion in the region 3.2, or the stationary phase σS subunits exhibited hypersensitivity to Lpm and increased cold sensitivity of RPo formation. Similar effects were produced by mutation located ~60Å away from the Lpm binding site within σ70 region 1.2, controlling −10 promoter element recognition. Our data suggested that template strand single-stranded DNA competes with Lpm for binding to RNAP and that σ70 regions 1.2 and 3.2 attenuate Lpm action by promoting DNA duplex opening.
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