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Programmed cell death in bacteria: translational repression by mRNA end-pairing
Abstract:
The hok/sok and pnd systems of plasmids R1 and R483 mediate plasmid maintenance by killing plasmid-free cells. Translation of the exceptionally stable hok and pnd mRNAs is repressed by unstable antisense RNAs. The different stabilities of the killer mRNAs and their cognate repressors explain the onset of translation in plasmid-free cells. The full-length hok and pnd mRNAs are inert with respect to translation and antisense RNA binding. We have previously shown that the mRNAs contain two negative translational control elements. Thus, the mRNAs contain upstream anti-Shine-Dalgarno elements that repress translation by shielding the Shine-Dalgarno elements. The mRNAs also contain fold-back-inhibition elements (fbi) at their 3' ends that are required to maintain the inert mRNA configuration. Using genetic complementation, we show that the 3' fbi elements pair with the very 5' ends of the mRNAs. This pairing sets the low rate of 3' exonucleolytical processing, which is required for the accumulation of an activatable pool of mRNA. Unexpectedly, the hok and pnd mRNAs were found to contain translational activators at their 5' ends (termed tac). Thus, the fbi elements inhibit translation of the full-length mRNAs by sequestration of the tac elements. The fbi elements are removed by 3' exonucleolytical processing. Mutational analyses indicate that the 3' processing triggers refolding of the mRNA 5' ends into translatable configurations in which the 5' tac elements base pair with the anti-Shine-Dalgarno sequences.
Insights
Plasmid maintenance systems use stable killer mRNAs (hok/pnd) and unstable antisense RNAs. Fold-back inhibition (fbi) elements sequester translational activators (tac) in inert mRNAs until 3' processing removes fbi, enabling translation.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Plasmid maintenance relies on toxic gene expression systems like hok/sok and pnd.
- These systems utilize stable killer mRNAs and unstable antisense RNAs for regulation.
- Previous work identified negative translational control elements in hok and pnd mRNAs.
Purpose of the Study:
- To elucidate the regulatory mechanisms of hok and pnd mRNA translation.
- To investigate the role of 3' fold-back inhibition (fbi) elements in mRNA regulation.
- To identify and characterize translational activators (tac) at the 5' end of these mRNAs.
Main Methods:
- Genetic complementation assays to study mRNA-protein interactions.
- Mutational analyses to investigate the function of fbi and tac elements.
- 3' exonucleolytical processing assays to determine processing rates and effects.
Main Results:
- 3' fbi elements pair with 5' mRNA ends, maintaining an inert configuration and low processing rate.
- Translational activators (tac) at the 5' end are sequestered by fbi elements in full-length mRNAs.
- 3' processing removes fbi elements, triggering 5' end refolding and enabling translation by unmasking tac elements.
Conclusions:
- The hok and pnd mRNA regulatory system involves a novel mechanism of translational control.
- 3' processing is crucial for activating translation by removing inhibitory elements and facilitating mRNA refolding.
- This intricate regulation ensures efficient plasmid maintenance through controlled expression of toxic genes.