Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3′-end processing.

Thisted, Thomas; Nielsen, Allan K.; Gerdes, Kenn

doi:10.1002/j.1460-2075.1994.tb06464.x

Cited by 62 publications

(85 citation statements)

References 31 publications

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“…Previously, we found that gel-purified truncated hok mRNA was translated efficiently, whereas full-length hok mRNA-1 and -2 were translated poorly (25,26,28). Here we expand our analyses using native RNAs that were not denatured and renatured before being subjected to translation (Fig.…”

Section: Fig 7 Coupled In Vitro Transcription/rnase H Cleavage Assaymentioning

confidence: 88%

“…Cleavage patterns were analyzed by primer extension using the hokA primer, 5Ј-GTGGTAGTTTCATGGC, according to Ref. 28.…”

Section: Methodsmentioning

confidence: 99%

“…2B. The 5Ј-to-3Ј base pairing in the full-length molecules yields compact structures, and hok mRNA-1 and -2 are inactive with respect to translation and antisense RNA binding (25)(26)(27)(28). Translation of hok mRNA-2 is activated by slow 3Ј-exonucleolytical removal of the fbi (fold-back inhibition) element (Fig.…”

mentioning

confidence: 99%

“…2B). The 3Ј processing releases the 5Ј tac (translational activator) element and thereby triggers a series of structural rearrangements prerequisite for translation and rapid Sok RNA binding (27)(28)(29). Thus, the activated refolded hok mRNA contains the energyrich tac stem and the antisense RNA target hairpin (Fig.…”

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confidence: 99%

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Temporal Translational Control by a Metastable RNA Structure

Møller‐Jensen

Franch

Gerdes

2001

Journal of Biological Chemistry

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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 posttranscriptional control mechanism.RNA molecules fold into highly ordered structures essential to their diverse biological functions. Accordingly, the question of how the linear sequence of ribonucleotides dictates the overall folding of an RNA has received much attention (1-6). Folding of RNA, whether occurring from a denatured state or sequentially in concomitance with its synthesis, involves the formation of a number of hierarchically ordered intramolecular interactions leading to increasing levels of structural organization. In both cases, structural rearrangements of kinetically favored folding intermediates may occur before the thermodynamically most stable conformation is reached (7,8). In the course of folding, RNA molecules face the risk of being trapped in nonequilibrium conformations. Because of the high thermodynamic stability of RNA secondary structures, rearrangement of such nonequilibrium conformations can constitute a substantial energy barrier, thus leading to kinetic trapping of the RNA in thermodynamically suboptimal conformations termed metastable structures (9 -11). Metastable folding intermediates have proved important to a number of biological processes including plasmid replication (12), replication of RNA by Q␤ replicase (13, 14), human immunodeficiency virus-1 RNA export to the cytoplasm (15), ribozyme activity (16 -20), and viroid replication (21-23).The hok/sok locus of plasmid R1 mediates plasmid maintenance by the killing of plasmid-free cells, also termed postsegregational killing (PSK) 1 (24). The PSK mechanism, which restricts synthesis of Hok toxin to newborn plasmid-free cells, is controlled entirely at the post-transcriptional level. The hok/ sok locus, presented schematically in Fig. 1, specifies two transcripts: the toxin-encoding hok (host killing) mRNA and the labile antisense inhibitor Sok RN...

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Section: Fig 7 Coupled In Vitro Transcription/rnase H Cleavage Assaymentioning

confidence: 88%

“…Cleavage patterns were analyzed by primer extension using the hokA primer, 5Ј-GTGGTAGTTTCATGGC, according to Ref. 28.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Temporal Translational Control by a Metastable RNA Structure

Møller‐Jensen

Franch

Gerdes

2001

Journal of Biological Chemistry

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“…However, there has been no mechanism shown to operate in prokaryotes that can remove an antisense RNA from its target (Thisted et al, 1994a). It has been shown that the hok mRNA is specifically cleaved at its 3'-end and that this processing leads to the translational activation of hok mRNA (Thisted et al, 1994a, b ;.…”

Section: Systems In Which the Killer Is A Protein The Synthesis Of Wmentioning

confidence: 99%

Conditionally lethal genes associated with bacterial plasmids

Holčı́k

Iyer

1997

Microbiology

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Suicidal genetically engineered microorganisms for bioremediation: Need and perspectives

2005

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In the past few decades, increased awareness of environmental pollution has led to the exploitation of microbial metabolic potential in the construction of several genetically engineered microorganisms (GEMs) for bioremediation purposes. At the same time, environmental concerns and regulatory constraints have limited the in situ application of GEMs, the ultimate objective behind their development. In order to address the anticipated risks due to the uncontrolled survival/dispersal of GEMs or recombinant plasmids into the environment, some attempts have been made to construct systems that would contain the released organisms. This article discusses the designing of safer genetically engineered organisms for environmental release with specific emphasis on the use of bacterial plasmid addiction systems to limit their survival thus minimizing the anticipated risk. We also conceptualize a novel strategy to construct "Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring/combining the knowledge of different plasmid addiction systems (such as antisense RNA-regulated plasmid addiction, proteic plasmid addiction etc.) and inducible degradative operons of bacteria.

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Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3′-end processing.

Cited by 62 publications

References 31 publications

Temporal Translational Control by a Metastable RNA Structure

Temporal Translational Control by a Metastable RNA Structure

Conditionally lethal genes associated with bacterial plasmids

Suicidal genetically engineered microorganisms for bioremediation: Need and perspectives

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