Regions 1.2 and 3.2 of the RNA Polymerase σ Subunit Promote DNA Melting and Attenuate Action of the Antibiotic Lipiarmycin.

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 σ(70), with a deletion in the region 3.2, or the stationary phase σ(S) subunits exhibited hypersensitivity to Lpm and increased cold sensitivity of RPo formation. Similar effects were produced by mutation located ~60 Å away from the Lpm binding site within σ(70) 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 σ(70) regions 1.2 and 3.2 attenuate Lpm action by promoting DNA duplex opening.