2 AMINO-ACIDS IN AN RNA POLYMERASE-SIGMA 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.