Coactivation of the RpoS-dependent proP P2 promoter by Fis and cyclic AMP receptor protein

The Escherichia coli proP P2 promoter, which directs the expression of an i ntegral membrane transporter of proline, glycine betaine, and other osmopro tecting compounds, is induced upon entry into stationary phase to protect c ells from osmotic shock. Transcription from the P2 promoter is completely d ependent on RpoS (sigma(38)) and Fis. Pis activates transcription by bindin g to a site centered at -41, which overlaps the promoter, where it makes a specific contact with the C-terminal domain of the alpha subunit of RNA pol ymerase (alpha-CTD). We show here that Fis and cyclic AMP (cAMP) receptor p rotein (CRP)-cAMP collaborate to activate transcription synergistically in vitro. Coactivation both in vivo and in vitro is dependent on CRP binding t o a site centered at -121.5, but CRP without Fis provides little activation . The contribution by CRP requires the correct helical phasing of the CRP s ite and a functional activation region 1 on CRP. We provide evidence that c oactivation is achieved by Pis and CRP independently contacting each of the two alpha-CTDs. Efficient transcription in vitro requires that both activa tors must be preincubated with the DNA prior to addition of RNA polymerase.