Role of activator site position and a distal UP-element half-site for sigma factor selectivity at a CRP/H-NS-activated sigma(S)-dependent promoter inEscherichia coli

Transcription initiation by the stress-associated sigma (S)-containing RNA polymerase holoenzyme (E sigma (S)) in Escherichia coli is often subject to complex regulation that involves multiple additional regulators and histon e-like proteins. csiD is a stationary phase-inducible sigma (S)-dependent g ene in E. coli that requires activation by cAMP-CRP (bound to a site centre d at -68.5 nucleotides upstream of the transcriptional start site) and is p ositively modulated by the abundant nucleoid-associated proteins H-NS and L rp. 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 cla ssic class I promoters; (ii) E sigma (S) prefers a CRP box location at -68. 5/-70.5, whereas E sigma (70) 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 d istal UP-element half-site located downstream of the CRP box, as demonstrat ed by mutational studies, in which this element was either eliminated or co mpleted to a full UP-element. The UP-element half-site favours E sigma (S)- mediated expression, whereas with the full UP-element, nearly wild-type lev els of csiD transcription were observed in the absence of sigma (S). Finall y, we show that the two histone-like proteins, H-NS and Lrp, both act by in fluencing activation by cAMP-CRP, but do so by different mechanisms. In par ticular, H-NS directly or indirectly increases positional stringency for th e CRP binding site. The implications of these findings with respect to sigm a factor selectivity, activation mechanisms used by the two holoenzymes and the architecture of sigma (S)-dependent promoters are discussed.