SIGMA-FACTOR MUTATIONS AFFECTING THE SEQUENCE-SELECTIVE INTERACTION OF RNA-POLYMERASE WITH -10-REGION SINGLE-STRANDED-DNA

The sigma subunit of RNA polymerase determines promoter recognition an d catalyzes DNA strand separation, The -35 promoter region is recogniz ed by a helix-turn-helix motif in region 4, while the -10 region is sp ecified, at least in part, by an amphipathic helix in region 2, We hav e proposed that conserved aromatic residues in sigma region 2.3 intera ct with the non-template strand of the -10 element to drive open compl ex formation, We now report that Bacillus subtilis sigma(A) holoenzyme , but neither core nor sigma(A) alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus seq uence, UV irradiation of holoenzyme-ssDNA complexes efficiently crossl inks sigma(A) to DNA and protease mapping supports a primary contact s ite in or near region 2, Several mutations in sigma(A) region 2.3, sho wn previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the effi ciency of photocrosslinking, These results support a model in which th ese aromatic amino acids are juxtaposed to ssDNA, consistent with thei r demonstrated role in stabilizing the open complex.