Organization of open complexes at Escherichia coli promoters - Location ofpromoter DNA sites close to region 2.5 of the sigma(70) subunit of RNA polymerase

A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma(70) in transcriptionally competent complexes betwee n Escherichia coli RNA polymerase and promoters. In this study we have engi neered sigma(70) to introduce a unique cysteine residue at a number of posi tions in region 2.5. Mutant proteins were purified, and in each case, the s ingle cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA . Fe (FeBABE). RNA polymerase co re reconstituted with tagged a derivatives was shown to be transcriptionall y active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE w as 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 ups tream of the -10 hexamer. This positioning is independent of promoter seque nce. A model for the interaction of this region of a with promoter DNA is d iscussed.