The σs subunit of RNA polymerase (RNAP) is the master regulator of the general stress response in Escherichia coli. Nevertheless, the selectivity of promoter recognition by the housekeeping σ70-containing and σ5-containing RNAP holoenzymes (Eσ70 and Eσs respectively) is not yet fully clarified, as they both recognize nearly identical −35 and −10 promoter consensus sequences. In this study, we show that in a subset of promoters, Eσs favours the presence of a distal UP-element half-site, and at the same time is unable to take advantage of a proximal half-site or a full UP-element. This is reflected by the frequent occurrence of distal UP-element half-sites in natural σs-dependent promoters and the absence of proximal half-sites. Eσ70, however, exhibits the opposite preference. The presence of the −35 element is a prerequisite for this differential behaviour. In the absence of the −35 element, half or full UP-element sites play no role in sigma selectivity, but the distal subsite leads to an equivalent, if not greater, transcriptional stimulation than the proximal one for both sigma factors. Finally, experiments using single amino acid substitutions of σs indicate that the foundation for this preference lies in an inability of σs to interact with the a subunit C-terminal domain.