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Metastable structures and refolding kinetics in hok mRNA of plasmid R1
J H Nagel1, A P Gultyaev, K Gerdes
1Leiden Institute of Chemistry, Gorlaeus Laboratories, The Netherlands.
Abstract:
Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.
Insights
Programmed cell death systems like hok/sok and pnd/pndB ensure plasmid maintenance. Metastable mRNA structures prevent premature toxin translation during transcription, stabilizing the system.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Plasmid maintenance relies on programmed cell death systems (e.g., hok/sok, pnd/pndB) that eliminate plasmid-free cells.
- Premature toxin translation during transcription is hypothesized to be prevented by 5' mRNA metastable hairpins.
Purpose of the Study:
- To provide experimental evidence for metastable structures in the 5' leader of hok and pnd mRNAs.
- To investigate the kinetics of refolding for these metastable structures in vitro.
- To assess the role of these structures in preventing premature translation and antisense RNA binding.
Main Methods:
- In vitro analysis of mRNA fragments from the 5' ends of hok and pnd genes.
- Kinetic studies to estimate refolding rates from metastable to stable structures.
- Structural analysis of mRNA leader regions.
Main Results:
- Experimental evidence confirms the existence of metastable hairpin structures in the 5' leader of hok and pnd mRNAs.
- Refolding kinetics of isolated mRNA fragments were estimated, supporting predicted structural rearrangements during transcription.
- Slow refolding rates allow for downstream hairpin formation, stabilizing the metastable structures.
Conclusions:
- The kinetic refolding parameters of hok and pnd mRNAs are consistent with their proposed role in preventing premature translation.
- Metastable mRNA structures act as a regulatory mechanism during transcription, controlling toxin expression and antisense RNA interaction.
- This mechanism is crucial for effective plasmid maintenance in bacterial systems.