Influence of the location of the cAMP receptor protein binding site on the geometry of a transcriptional activation complex in Escherichia coli

Patrick Eichenberger, Sylvie Déthiollaz, Nobuyuki Fujita, Akira Ishihama, Johannes Geiselmann

Research output: Contribution to journalArticlepeer-review

Abstract

The interactions between the cAMP receptor protein (CRP) and RNA polymerase during transcriptional activation at the Escherichia colt malt promoter have been analyzed using a combination of footprinting methods. We show that a closed complex is formed at this promoter in the absence of activator and that CRP merely stabilizes the open complex. The α-subunits of the RNA polymerase are involved in this effect as shown by KMnO4 footprinting. The open complex formed in the presence of CRP is structurally identical to the one found at a CRP-independent promoter up-mutant. UV-laser footprinting yields distinct signals for the different protein-DNA interactions within the complex and for interactions between CRP and RNA polymerase. We monitor these signals in promoter variants that place the CRP binding site at different distances upstream of the start site of transcription. Signals within the core promoter region, as well us those located just upstream of the -35 hexamer, are unaffected by the position of the CRP binding site. Contacts of RNA polymerase with the upstream promoter region change in a mutant RNA polymerase containing a truncated α-subunit. We conclude that at least one of the α-subunits of RNA polymerase binds to DNA upstream of the -35 hexamer and that this interaction is unaffected by the position of the CRP binding site. We discuss models that account for the different activities of CRP in transcriptional activation as a function of promoter geometry.

Original languageEnglish (US)
Pages (from-to)15302-15312
Number of pages11
JournalBiochemistry
Volume35
Issue number48
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry

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