Abstract
Programmed cell death by the hok/sok locus of plasmid R1 relies on a complex translational control mechanism. The highly stable hok mRNA is activated by 3'-end exonucleolytical processing. Removal of the mRNA 3' end releases a 5'-end sequence that triggers refolding of the mRNA. The refolded hok mRNA is translatable but can also bind the inhibitory Sok antisense RNA. Binding of Sok RNA leads to irreversible mRNA inactivation by an RNase III-dependent mechanism. A coherent model predicts that during transcription hok mRNA must be refractory to translation and antisense RNA binding. Here we provide genetic evidence for the existence of a 5' metastable structure in hok mRNA that locks the nascent transcript in an inactive configuration in vivo. Consistently, the metastable structure reduces the rate of Sok RNA binding and completely blocks hok translation in vitro. Structural analyses of native RNAs strongly support that the 5' metastable structure exists in the nascent transcript. Further structural analyses reveal that the mRNA 3' end triggers refolding of the mRNA 5' end into the more stable tac-stem conformation. These results provide a profound understanding of an unusual and intricate post-transcriptional control mechanism.
Highlights
RNA molecules fold into highly ordered structures essential to their diverse biological functions
The U21A Mutation in hok mRNA Increases Sok Antisense RNA Binding in Vivo—Based on folding simulations and phylogenetic analyses of the hok family of mRNAs, two hairpin structures were proposed to form at the hok mRNA 5Ј end [30]
Along with the U21A mutation, we introduced the second-site A9U mutation, which restores the possibility of forming the metastable hairpin
Summary
Further structural analyses reveal that the mRNA 3 end triggers refolding of the mRNA 5 end into the more stable tac-stem conformation These results provide a profound understanding of an unusual and intricate posttranscriptional control mechanism. The activated refolded hok mRNA contains the energyrich tac stem and the antisense RNA target hairpin (Fig. 2C), the latter of which is required both for translation and rapid antisense RNA binding [25, 26, 29, 30]. We present evidence that hok mRNA specifies a structure that simultaneously prevents antisense RNA binding and synthesis of Hok toxin during transcription. This molecular safeguard consists of two small metastable hairpins at the mRNA 5Ј end (Fig. 2A).
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