Role of activator site position and a distal UP‐element half‐site for sigma factor selectivity at a CRP/H‐NS‐activated σS‐dependent promoter in Escherichia coli

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Abstract
Transcription initiation by the stress-associated σS-containing RNA polymerase holoenzyme (EσS) in Escherichia coli is often subject to complex regulation that involves multiple additional regulators and histone-like proteins. csiD is a stationary phase-inducible σS-dependent gene in E. coli that requires activation by cAMP-CRP (bound to a site centred at −68.5 nucleotides upstream of the transcriptional start site) and is positively modulated by the abundant nucleoid-associated proteins H-NS and Lrp. By shifting the CRP box to positions between −80.5 and −60.5, we could demonstrate that: (i) activation is equally helix phase dependent as at classic class I promoters; (ii) EσS prefers a CRP box location at −68.5/−70.5, whereas Eσ70 is nearly inactive with such an arrangement; and (iii) with the CRP site moved to −60.5, transcription can be initiated efficiently by both holoenzymes. The csiD promoter region also contains a distal UP-element half-site located downstream of the CRP box, as demonstrated by mutational studies, in which this element was either eliminated or completed to a full UP-element. The UP-element half-site favours EσS-mediated expression, whereas with the full UP-element, nearly wild-type levels of csiD transcription were observed in the absence of σS. Finally, we show that the two histone-like proteins, H-NS and Lrp, both act by influencing activation by cAMP-CRP, but do so by different mechanisms. In particular, H-NS directly or indirectly increases positional stringency for the CRP binding site. The implications of these findings with respect to sigma factor selectivity, activation mechanisms used by the two holoenzymes and the architecture of σS-dependent promoters are discussed.

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