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Related Experiment Videos

Characterization of two hypertransposing Tn5 mutants.

T W Wiegand1, W S Reznikoff

  • 1Department of Biochemistry, University of Wisconsin-Madison 53706.

Journal of Bacteriology
|February 1, 1992
PubMed
Summary
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Mutant transposase proteins (Tnp) in Escherichia coli enhance Tn5 transposition frequency and activity. These EK transposase mutants alter interactions with DNA, suggesting a dual role for transposase in regulating transposition.

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Tn5 transposition in Escherichia coli is regulated by transposase (Tnp) and inhibitor (Inh) proteins.
  • Transposase typically promotes transposition in cis, while the inhibitor acts in trans.

Purpose of the Study:

  • To investigate the function of specific transposase mutants (EK110 and EK345) in Tn5 transposition.
  • To elucidate the regulatory mechanisms of Tn5 transposition involving transposase species.

Main Methods:

  • Isolation and characterization of two glutamate-to-lysine transposase mutants (EK110 and EK345).
  • Gel retardation assays to analyze protein-DNA interactions.
  • Transposition frequency assays in cis and trans.

Main Results:

Related Experiment Videos

  • EK transposase mutants increased Tn5 transposition frequency 6- to 16-fold in cis and enhanced trans activity.
  • Mutant transposases formed novel complexes with transposon DNA compared to wild-type.
  • Wild-type transposase exhibited self-inhibition, which was reduced in EK mutants.

Conclusions:

  • A model proposing two transposase species (cis-activating and trans-inhibiting) is presented.
  • EK transposase mutants shift the ratio towards the cis-activating species, explaining their phenotype.
  • This study reveals a novel self-regulatory function of transposase in Tn5 transposition.