Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: Two translational control elements in the pnd mRNA

Programmed cell death by hok/sok of plasmid R1: Processing at the hok mRNA 3′-end triggers structural rearrangements that allow translation and antisense RNA binding

“…The ucb element is shown in red in Figure 2. Previously we suggested that pairing between SD mok and ucb prevents ribosome entry at SD mok and thereby inhibits translation of mok (and hok; Nielsen & Gerdes, 1995). A nearly perfect repeat of the 9 nt ucb element is located 58 nt downstream of ucb (Figure 2).…”

“…The mutation shown in Figure 6 was introduced in the hok/sok system by double PCR as described by Nielsen & Gerdes (1995). PCR was performed using the pBR322 vector carrying the 580 bp wild-type hok/sok system, inserted in the EcoRI -BamHI sites, as template and the pBR322 EcoRI CW and pBR322 BamHI CCW external primers.…”

Coupled nucleotide covariations reveal dynamic RNA interaction patterns

“…A unique example of transitions between alternative RNA structures is provided by the family of genes that mediate stabilization of plasmids by killing newborn plasmid-free cells (for reviews, see Gerdes et al+, 1990Gerdes et al+, , 1997)+ The prototype locus, hok/sok from plasmid R1, produces a stable toxin-encoding mRNA and an unstable (antidote) antisense RNA+ The Sok antisense RNA (suppression of killing) inhibits translation of the toxin hok mRNA (host killing)+ The postsegregational killing stabilizes the plasmids carrying the hok/ sok system, because the toxin encoded by the mRNA that remains in a cell even after the plasmid loss kills only plasmid-free cells whereas cells retaining the plasmid are rescued by the replenished Sok RNA molecules+ Activation of hok translation is regulated by alternative secondary structures in the hok mRNA translation initiation region+ One of these structures is both translationally active and competent in antisense RNA binding, whereas the other is inert in both respects (Franch et al+, 1997)+ Furthermore, the functionally important structural rearrangements turned out to be triggered by refoldings at the very ends of the mRNA+ In these regions, phylogenetic conservation of the specific RNA folding pathway (Gultyaev et al+, 1997) results in a unique pattern of coupled covariations that reflects three different functional conformations, present at different stages in the life cycle of the RNA (Fig+ 1A)+ The folding pathway works as a clock mechanism, keeping the toxin mRNA inactive for a considerable time while providing its activation at a proper moment+ During transcription, the metastable hairpin at the very 59 end (Fig+ 1A) induces formation of inactive folding in the translation initiation region, located further downstream in the sequence (not shown in the Fig+ 1A)+ Upon completion of transcription, the inactive mRNA is locked by pairing of the 59 end to a so-called fold-back-inhibition element (fbi ), located at the very 39 end+ The fbi interaction disrupts the metastable hairpin with the formation of the new stemloop structure in the 59-proximal region+ In this stable conformation, the full-length mRNA is neither translated nor does it bind the inhibitory antisense RNA+ This allows for the accumulation of a pool of inactive mRNAs ready to be activated if the plasmid-born gene is lost+ Translation of hok mRNA is activated by slow 39-end processing (Thisted et al+, 1994;Nielsen & Gerdes, 1995)+ The removal of the 39-terminal nucleotides, including the inhibitory fbi element, results in the third conformation at the 59 end, characterized by the extension of the stable stem (Fig+ 1A)+ This final rearrangement in the truncated RNA triggers the formation of the active structure downstream (not shown), which results either in translation or in Sok antisense RNA binding+ All three conformations, shown in Figure 1A, are vital for the mechanism and conserved in other hokhomologous gene systems (Gerdes et al+, 1997;Pedersen & Gerdes, 1999)+ The constituent stems are supported by covariations that are interdependent due to overlaps of mutually exclusive structures+ For example (Fig+ 1A), the covariation of pair 3-28 in the metastable hairpin (C-G in hok and U-A in related gene srnB ) is coupled with nt 394 (hok numbering) in the fbi element, which pairs to nt 3 in the fulllength RNA and is, correspondingly, G in hok and A in srnB+ Similarly, the covariation of pair 11-19 in the metastable h...…”

“…The folding pathway of hok-related mRNAs was predicted by RNA folding simulations using a genetic algorithm (Gultyaev et al+, 1995a)+ The folding pathway was supported by numerous covariations in all regulatory secondary structure elements (Gultyaev et al+, 1997)+ Furthermore, the predicted structures were verified experimentally by mutational analyses, structure probing, and functional assays using translation and antisense RNA binding (Thisted et al+, 1995;Nielsen & Gerdes, 1995;Franch & Gerdes, 1996;Franch et al+, 1997)+ Recently, the formation of the metastable hairpin (which, according to the model, folds only transiently), was studied in vitro by structure probing of RNA fragments from hok and pnd genes, captured in metastable state (Nagel et al+, 1999)+ This approach also allowed the observation of relatively slow kinetics of the hairpin unfolding, consistent with the assumption about its metastable character+…”

Endoribonuclease type II toxin–antitoxin systems: functional or selfish?

“…Plasmid TAs were shown to induce addiction to enforce cell stasis/ death of cells cured of TA-encoding plasmids during nonselective conditions, resulting in the increased prevalence of plasmids within a population [11,24,65,66]. Logically, if a bacterium were to be cured of a TA-encoding plasmid and yet survive peer pressure in the limiting conditions, it must have acquired an antidote for the toxin.…”