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The σS subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of EσS RNA polymers, using σS derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5). The chemical nuclease FeBABE can cleave DNA in proximity to the chelate. The overall cutting pattern of EσS open complexes does not depend on the nature of the promoter and is similar to that obtained with Eσ70, but extends towards the downstream part of the promoter. The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1. In contrast to σ70 region 2.1 of σS appears to be far from DNA. Region 4.2 of σS appears less accessible than its counterpart in σ70 and FeBABE positioned in the turn of the helix-turn-helix (HTH) motif in region 4.2 reacts only weakly with the −35 promoter element. This provides a structural basis for the minor role of the −35 sequence in σS-dependent promoter recognition. [ABSTRACT FROM AUTHOR]