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Directionality of DnaA protein/DNA interaction. Active orientation of the DnaA protein/dnaA box complex in

C Schaefer1, W Messer

  • 1Max-Planck-Institut für Molekulare Genetik, Berlin, FRG.

The EMBO Journal
|May 1, 1989
PubMed

Insights

The DnaA protein binds DNA and stops transcription, regulating its own gene expression. This autoregulation mechanism involves specific DNA sequences and strand orientation for termination.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biochemistry

Background:

  • The DnaA protein is a key initiator of DNA replication in bacteria.
  • Transcription and DNA replication processes can interfere with each other.
  • Autoregulation is a common mechanism for controlling gene expression.

Purpose of the Study:

  • To investigate the mechanism of DnaA protein-mediated transcription termination.
  • To determine the role of the dnaA box sequence and orientation in termination.
  • To elucidate the contribution of DnaA-dependent termination to dnaA gene autoregulation.

Main Methods:

  • Utilized a model system to monitor transcription rates using a reporter gene.
  • Assessed transcription termination efficiency relative to the dnaA box.
  • Employed oligonucleotide-directed mutagenesis to alter the dnaA box sequence within the dnaA gene.

Main Results:

  • DnaA protein binding to the dnaA box (5'-TTTTCCACA) effectively blocked transcribing RNA polymerase, causing transcription termination.
  • Termination occurred regardless of whether the dnaA box region was translated, indicating a direct physical blockage.
  • Only the specific orientation of the dnaA box on the non-coding strand mediated termination, suggesting single-strand DNA contact by DnaA.
  • Mutating the dnaA box within the dnaA coding region led to increased dnaA gene expression.

Conclusions:

  • DnaA protein binding to its consensus site (dnaA box) functions as a transcriptional roadblock.
  • DnaA-mediated transcription termination is orientation-dependent, implying specific DNA strand interaction.
  • This termination mechanism is a crucial component of the autoregulation of the dnaA gene.

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