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

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 -60 Å 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.