FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli

The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts. The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method. Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene. In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation. Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit. The alpha[L262A], alpha[R265A] and alpha[N268A] substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.