Altered promoter recognition by mutant forms of the σ70 subunit of Escherichia coli RNA polymerase

Abstract

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the σ 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of σ 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of σ 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the −35 region, and that a sequence internal to σ 70, in a region which is highly conserved among σ factors, recognizes the −10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the −35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effects on promoter recognition.