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Control of ColE1 plasmid replication. Interaction of Rom protein with an unstable complex formed by RNA I and RNA II

J Tomizawa1

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

Journal of Molecular Biology
|April 20, 1990
PubMed

Insights

The plasmid-specified protein Rom enhances the binding of RNA I to RNA II, a crucial step in ColE1 plasmid replication. Rom stabilizes the RNA-RNA complex, creating a more efficient pathway for replication inhibition.

Area of Science:

  • Molecular Biology
  • Plasmid Replication
  • RNA-RNA Interactions

Background:

  • ColE1 plasmid replication initiation is regulated by RNA I, which inhibits primer RNA (RNA II) formation.
  • The plasmid-encoded protein Rom modulates this regulatory process.

Purpose of the Study:

  • To investigate the mechanism by which the Rom protein affects the interaction between RNA I and RNA II.
  • To elucidate the role of Rom in stabilizing the RNA I-RNA II complex.

Main Methods:

  • In vitro binding assays to study RNA I and RNA II complex formation.
  • Enzyme sensitivity assays (RNase) to detect stable RNA-RNA complexes.
  • Kinetic analysis of complex formation and interconversion in the presence and absence of Rom.

Main Results:

  • RNA I and RNA II form an unstable complex (C*) in vitro.
  • The Rom protein binds to C*, converting it to a more stable complex (Cm*).
  • Both C* and Cm* can further transform into more stable complexes (C** and Cm**, respectively), with Rom facilitating this stabilization.

Conclusions:

  • Rom protein stabilizes the interaction between RNA I and RNA II, thereby enhancing the inhibition of ColE1 plasmid replication.
  • Rom acts by converting an unstable intermediate complex into a more stable form, establishing an alternative pathway for stable RNA-RNA binding.

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