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Related Experiment Videos

Sequence-specific interactions between promoter DNA and the RNA polymerase sigma factor E

K M Tatti1, M F Shuler, C P Moran

  • 1Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Journal of Molecular Biology
|October 13, 1995
PubMed
Summary

Specific amino acid residues in sigma E from Bacillus subtilis control promoter recognition. Substitutions at positions 217 and 219 alter promoter specificity, revealing key interactions within the -35 region.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Bacterial transcription relies on sigma factors binding to promoter regions.
  • Sigma E and sigma K are secondary sigma factors in Bacillus subtilis with distinct promoter specificities.
  • Understanding sigma factor-promoter interactions is crucial for regulating gene expression.

Purpose of the Study:

  • To identify amino acid residues in sigma E responsible for -35 promoter region recognition.
  • To investigate how specific mutations in sigma E affect its promoter specificity.
  • To elucidate the molecular basis of sigma factor-DNA interactions.

Main Methods:

  • Site-directed mutagenesis of the Bacillus subtilis sigma E gene.
  • In vitro transcription assays using wild-type and mutant sigma factors.

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  • Analysis of promoter activity with native and modified promoter sequences.

    • Main Results:

    • A single amino acid substitution at position 217 in sigma E conferred recognition of sigma K-dependent promoters.
    • This substitution specifically suppressed a base-pair mutation at position -31 in the spoIIID promoter.
    • Residue 219 in sigma E influenced interaction specificity with position -33 of the spoIIID promoter.

    Conclusions:

    • Amino acid residues at positions 217 and 219 of sigma E are critical determinants of -35 promoter region specificity.
    • These residues likely form direct contacts with specific base pairs in the promoter -35 region.
    • Mutational analysis provides insight into the molecular mechanisms of sigma factor-DNA binding and specificity.