Escherichia coli promoter opening and -10 recognition: mutational analysis of sigma70.

The opening of specific segments of DNA is required for most types of genetic readout, including sigma70-dependent transcription. To learn how this occurs, a series of single point mutations were introduced into sigma70 region 2. These were assayed for duplex DNA binding, DNA opening and DNA double strand-single strand fork junction binding. Band shift assays for closed complex formation implicated a series of arginine and aromatic residues within a minimal 26 amino acid region. Permanganate assays implicated two additional aromatic residues in DNA opening, known to form a parallel stack of the type that can accept a flipped-out base. Substitution for either of these aromatics had no effect on duplex probe recognition. However, when a single unpaired -11 nucleotide is added to the probe, the mutants fail to bind appropriately to give heparin resistance. A model for DNA opening is presented in which duplex recognition by regions 2.3, 2.4 and 2.5 of sigma positions the pair of aromatic amino acids, which then create the fork junction required for stable opening.