σ factor mutations affecting the sequence-selective interaction of RNA polymerase with -10 region single-stranded DNA

Abstract

The σ subunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation. The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2. We have proposed that conserved aromatic residues in σ region 2.3 interact with the non-template strand of the -10 element to drive open complex formation. We now report that Bacillus subtilis σ(A) holoenzyme, but neither core nor σ(A) alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence. UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks σ(A) to DNA and protease mapping supports a primary contact site in or near region 2. Several mutations in σ(A) region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking. These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex.