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Related Experiment Videos

Quantitation of ColE1-encoded replication elements.

M Brenner1, J Tomizawa

  • 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.

Proceedings of the National Academy of Sciences of the United States of America
|January 15, 1991
PubMed
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Replication of Escherichia coli plasmid ColE1 relies on RNA primers. The plasmid copy number is mainly controlled by RNA I synthesis, not RNA II, according to in vivo measurements of key molecules.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Biochemistry

Background:

  • Escherichia coli plasmid ColE1 replication initiates from an RNA primer.
  • ColE1 RNA II forms the primer by hybridizing to the DNA template.
  • RNA I binding to RNA II inhibits primer formation, a process regulated by the Rom protein.

Purpose of the Study:

  • To quantitatively analyze the regulation of ColE1 plasmid synthesis.
  • To determine the in vivo levels and half-lives of RNA I, RNA II, and Rom protein.

Main Methods:

  • In vivo measurement of intracellular concentrations of RNA I, RNA II, and Rom protein.
  • Determination of RNA half-lives in Escherichia coli.

Main Results:

Related Experiment Videos

  • Intracellular concentrations were approximately 1 microM for RNA I, 7 nM for RNA II, and 1 microM for Rom protein.
  • Both RNA I and RNA II exhibited half-lives of approximately 2 minutes.
  • A quantitative model indicated plasmid copy number is highly sensitive to RNA I synthesis rate.

    • Conclusions:

    • The rate of RNA II synthesis has minimal impact on plasmid copy number.
    • Plasmid copy number regulation is primarily dictated by the synthesis rate of RNA I.