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

Didier Nègre, Christelle Oudot, Jean François Prost, Katsuhiko Murakami, Akira Ishihama, Alain J. Cozzone, Jean Claude Cortay

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Abstract

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°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 the C-terminal domain of their α subunit. The α[L262A], α[R265A] and α[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.

Original languageEnglish (US)
Pages (from-to)355-365
Number of pages11
JournalJournal of Molecular Biology
Volume276
Issue number2
DOIs
StatePublished - Feb 20 1998

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

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    Nègre, D., Oudot, C., Prost, J. F., Murakami, K., Ishihama, A., Cozzone, A. J., & Cortay, J. C. (1998). FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli. Journal of Molecular Biology, 276(2), 355-365. https://doi.org/10.1006/jmbi.1997.1548