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The influence of a metastable structure in plasmid primer RNA on antisense RNA binding kinetics
A P Gultyaev1, F H van Batenburg, C W Pleij
1Leiden Institute of Chemistry, Leiden University, The Netherlands.
Abstract:
Replication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II conformations, formed during the transcription, and effectively inhibits the primer function within some time interval. In this paper, the folding pathways for the wild type and copy number mutants of ColE1 RNA II are studied using simulations by a genetic algorithm. The simulated pathways reveal a transient formation of a metastable structure, which is stabilized by copy number mutations. The folding kinetics of the proposed conformational transitions is calculated using a model of a multistep refolding process with elementary steps of double-helical stem formation or disruption. The approximation shows that the lifetime of the metastable structure is relatively long and is considerably increased in the mutants, resulting in a delay of the formation of the stable RNA II structure, which is the most sensitive to the inhibition by the antisense RNA I. Thus the effect of copy number mutations can be interpreted as a compression of the time window of effective inhibition due to an increased time spent by the RNA II in the metastable state. The implications of metastable foldings in RNA functioning are discussed.
Insights
Copy number mutations in ColE1 RNA II alter its folding pathway, stabilizing a transient structure. This delays the formation of the active conformation, impacting plasmid replication control.
Area of Science:
- Molecular Biology
- Biophysics
Background:
- ColE1 plasmid replication is regulated by RNA II and RNA I interactions.
- RNA II exists in alternative conformations affecting primer function and inhibition by RNA I.
Purpose of the Study:
- Investigate the folding pathways of wild-type and mutant ColE1 RNA II.
- Understand how copy number mutations affect RNA II folding kinetics and stability.
Main Methods:
- Simulations using a genetic algorithm to study RNA II folding pathways.
- Kinetic modeling of multistep refolding processes.
Main Results:
- Identified a transient, metastable RNA II structure.
- Copy number mutations stabilize this metastable state, increasing its lifetime.
- This stabilization delays the formation of the stable RNA II structure, reducing inhibition by RNA I.
Conclusions:
- Metastable RNA II structures play a role in regulating plasmid replication.
- Copy number mutations modulate replication control by altering RNA II folding kinetics.
- The findings have implications for understanding RNA structure-function relationships.