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Arm-domain interactions in AraC

B Saviola1, R Seabold, R F Schleif

  • 1Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.

Journal of Molecular Biology
|June 20, 1998
PubMed
Summary
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Deletions in the N-terminal arm of the AraC protein cause constitutive activity of the pBAD promoter. This arm is crucial for maintaining the inactive state of the AraC regulator.

Area of Science:

  • * Molecular Biology
  • * Genetics
  • * Biochemistry

Background:

  • * The AraC protein regulates the l-arabinose operon in Escherichia coli.
  • * Understanding AraC's regulatory mechanism is key to controlling gene expression.

Purpose of the Study:

  • * To investigate the role of the N-terminal arm of the AraC protein in regulating the pBAD promoter.
  • * To identify specific regions within the AraC protein involved in transcriptional activation and repression.

Main Methods:

  • * Site-directed mutagenesis of the AraC protein's N-terminal region.
  • * Screening for constitutive and non-inducing mutants of the pBAD promoter.
  • * Analysis of arabinose binding affinity to mutated AraC proteins.
  • * Determination of crystal structures of the AraC N-terminal domain.

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Main Results:

  • * N-terminal deletions beyond the sixth amino acid resulted in constitutive pBAD promoter activity.
  • * Mutations in the N-terminal arm did not prevent transcription activation but were essential for repression.
  • * Mutations in the C-terminal domain affecting arabinose binding could be suppressed by N-terminal modifications.
  • * Crystal structures revealed that N-terminal arms bind to C-terminal DNA-binding domains in the absence of arabinose.

Conclusions:

  • * The N-terminal arm of AraC is not essential for transcription activation but plays a critical role in maintaining the repressed state.
  • * In the absence of arabinose, the N-terminal arms interact with C-terminal DNA-binding domains, inhibiting transcription.
  • * Arabinose binding causes a conformational change, releasing the N-terminal arms and allowing DNA binding and transcription activation.