Interaction of Escherichia coli RNA polymerase holoenzyme containing σ32 with heat shock promoters: DNase I footprinting and methylation protection

The DNase I protection pattern of Eσ32 was assayed on three heat shock promoters, the Eσ32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the Eσ32 promoter upstream from rpoD. Eσ32 protected each of these promoters from DNase I digestion from around −60 to around +20. Protection from dimethyl sulfate methylation was assayed at the groE promoter. Eσ32 binding altered the sensitivity to methylation of bases in the vicinity of both the −10 and −35 regions. The DNase I footprints for the Eσ32 promoters were very similar to the DNase I footprint of Eσ70 on the lacUV5 promoter. After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that Eσ32, like Eσ70, contacts the DNA primarily on one face of the helix in the −35 region and on both faces in the −10 region.