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The effect of promoter strength, supercoiling and secondary structure on mutation rates in Escherichia coli

Karen H Schmidt1, Jacqueline M Reimers, Barbara E Wright

  • 1Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.

Molecular Microbiology
|May 13, 2006
PubMed

Insights

This study engineered opal stop codon mutations into a chloramphenicol resistance gene. Increased transcription and altered DNA supercoiling significantly affected mutation reversion rates.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Opal stop codons (UGA) are critical in gene expression regulation.
  • Understanding factors influencing mutation reversion is key for genetic stability studies.

Purpose of the Study:

  • To investigate the impact of transcription levels and DNA supercoiling on the reversion frequency of specific mutations.
  • To analyze the correlation between predicted and experimentally determined mutation reversion rates.

Main Methods:

  • Engineered four distinct opal stop codon mutations into a chloramphenicol resistance (cat) gene.
  • Utilized the mfg computer program for predicting relative reversion frequencies.
  • Compared reversion rates under different promoter strengths (lac vs. tac) and supercoiling conditions (topA mutants).

Main Results:

  • Experimental reversion frequencies correlated with mfg predictions.
  • Replacing the lac promoter with the stronger tac promoter increased reversion rates 12- to 30-fold.
  • Increased negative supercoiling (topA mutant) elevated reversion rates, while decreased supercoiling (topA gyrB mutant) lowered them.

Conclusions:

  • Transcription levels and DNA supercoiling are significant modulators of mutation reversion rates.
  • The mfg program provides a reliable prediction of base mutability and reversion frequencies.
  • These findings have implications for understanding genetic instability and DNA repair mechanisms.

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