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The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the s 54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the s 54 -RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on s 54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of s 54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFDHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFDHTH from a plasmid was employed. Thus, the s 54 -dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed s 54 -RNAP-promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.