Two amino acids in an RNA polymerase σ factor involved in the recognition of adjacent base pairs in the -10 region of a cognate promoter

The recognition of promoter region -10 nucleotide sequences in prokaryotes is believed to be mediated by a segment of α-helix in a region of RNA polymerase σ factors called 2.4. Earlier genetic studies implicated Thr-100 in region 2.4 of the Bacillus subtilis σ factor σ(H) in the recognition of the G·C base pair at position -13 in the -10 region (GAAT) of a cognate promoter. In confirmation of this assignment, we now show that a change-of-specificity mutant of σ(H) in which Thr-100 was replaced with isoleucine suppresses a G·C → A·T nucleotide substitution at position -13 but no other 'promoter down mutations' (causing impaired promoter activity) at positions -13, -12, and -11. We also show that a loss-of-contact mutant created by the replacement of Thr-100 with alanine (having a short side chain) enables σ(H) to tolerate three different promoter down mutations at position -13 but not down mutations at other positions. Finally, we suggest the identification of an additional amino acid involved in base-pair recognition by the demonstration that the replacement of Arg-96 with alanine specifically suppresses an A·T → G·C promoter down mutation at position -12. The identification of amino acids that are four residues apart that are involved in the recognition of adjacent base pairs may fix the orientation of region 2.4 (its NH₂ terminus being proximal to the promoter transcription start site) and is consistent with a model in which the recognition of promoter region -10 nucleotide sequences is mediated by an α-helix in which residues involved in base-pair contact are separated by one turn and clustered on one face of the helix.