A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of σ70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered σ70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA · Fe (FeBABE). RNA polymerase core reconstituted with tagged σ derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of σ with promoter DNA is discussed.
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