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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.

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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:

  • 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.