Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure

Franch, Thomas; Petersen, Michael; Wagner, E. Gerhart H.; Jacobsen, Jens Peter; Gerdes, Kenn

doi:10.1006/jmbi.1999.3306

Cited by 151 publications

(132 citation statements)

References 53 publications

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“…2C). In contrast, Sok RNA bound rapidly to both purified wild-type and super-tac hok mRNAs, indicating that these molecules contain the antisense RNA target hairpin that mediates that highest rate of antisense binding (25,39). Note that the molecules used above were denatured and renatured during their purification.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

Temporal Translational Control by a Metastable RNA Structure

Møller‐Jensen

Franch

Gerdes

2001

Journal of Biological Chemistry

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“…The antisense and attenuator loops both have YUNR [Y-pyrimidine (C,U), R-purine (A,G), N-Y, or R] motifs, which are ubiquitous in recognition loops of natural antisense gene regulation systems (26,27), and have been used as a design element in synthetic RNA regulators (10). We therefore searched for mutations that preserved these motifs, while otherwise disrupting interac- .…”

Section: Independently Acting Attenuator Variants Can Be Engineered Tmentioning

confidence: 99%

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

Lucks

Mutalik

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

234

358

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The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.gene networks | regulatory systems | orthogonal regulators N oncoding RNA has been found to play a central role in regulating gene expression in both prokaryotes and eukaryotes. Recently, the diverse roles of RNA-mediated regulation have become important tools for synthetic biology applications ranging from detecting metabolic state (1), balancing metabolic pathway expression (2), tightly regulating toxin genes (3), and detecting environmentally harmful chemicals (4). In particular, RNA-based genetic parts have been engineered that regulate transcription through RNA-mediated transcription factor recruitment (5, 6), transcript stability through small-molecule-mediated ribozyme cleavage (1, 7) and siRNA targeted degradation (8), and translation through cis-acting mRNA conformational changes (9) and trans-acting antisense RNA-mRNA interactions (10,11).This wide array of RNA function is beginning to be used to engineer programmable genetic circuitry required for the next level of synthetic biology applications (12). By interfacing with protein-based transcription factors and repressors, hybrid RNA∕ protein cascades have been made that perform sophisticated logic evaluation (8) and even count extracellular events (13). Much like previous work on protein-based cascades (14), protein regulators propagate the signal between different levels of the hybrid cascades. This makes the inner workings of these cascades complicated by the many interconversions between mRNA and protein that must take place. This not only incre...

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“…All elements of the model depicted in figure 3b were verified experimentally [52,60,61,65]. A metastable hairpin forms in the nascent transcript at the very 5 0 -end of hok mRNA (figure 3b(i)) and persists until the mRNA 5 0 -end pairs with the mRNA 3 0 -end in the full-length (FL) mRNA to form a highly folded, stable structure ( figure 3b(ii)).…”

Section: Complex Regulation Of the Hok/sok Module Of Plasmid R1mentioning

confidence: 84%

“…Removal of 40 nt at the 3 0 end of FL hok mRNA disrupts the fbi -tac interaction and triggers refolding into the truncated, active version of the mRNA that can either bind the antisense or be translated. Everted bases indicated with dashes symbolize the U-turn loop structure present in the refolded hok mRNA, which functions as the initial recognition element for antisense RNA binding and thereby increases the recognition reaction approximately 10-fold [52]. SD, Shine and Dalgarno elements.…”

Section: Complex Regulation Of the Hok/sok Module Of Plasmid R1mentioning

confidence: 99%

Hypothesis: type I toxin–antitoxin genes enter the persistence field—a feedback mechanism explaining membrane homoeostasis

Gerdes

2016

Phil. Trans. R. Soc. B

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Bacteria form persisters, cells that are tolerant to multiple antibiotics and other types of environmental stress. Persister formation can be induced either stochastically in single cells of a growing bacterial ensemble, or by environmental stresses, such as nutrient starvation, in a subpopulation of cells. In many cases, the molecular mechanisms underlying persistence are still unknown. However, there is growing evidence that, in enterobacteria, both stochastically and environmentally induced persistence are controlled by the second messenger (p)ppGpp. For example, the ‘alarmone’ (p)ppGpp activates Lon, which, in turn, activates type II toxin–antitoxin (TA) modules to thereby induce persistence. Recently, it has been shown that a type I TA module, hokB / sokB , also can induce persistence. In this case, the underlying mechanism depends on the universally conserved GTPase Obg and, surprisingly, also (p)ppGpp. In the presence of (p)ppGpp, Obg stimulates hokB transcription and induces persistence. HokB toxin expression is under both negative and positive control: SokB antisense RNA inhibits hokB mRNA translation, while (p)ppGpp and Obg together stimulate hokB transcription. HokB is a small toxic membrane protein that, when produced in modest amounts, leads to membrane depolarization, cell stasis and persistence. By contrast, overexpression of HokB disrupts the membrane potential and kills the cell. These observations raise the question of how expression of HokB is regulated. Here, I propose a homoeostatic control mechanism that couples HokB expression to the membrane-bound RNase E that degrades and inactivates SokB antisense RNA. This article is part of the themed issue ‘The new bacteriology’.

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Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure

Cited by 151 publications

References 53 publications

Temporal Translational Control by a Metastable RNA Structure

Temporal Translational Control by a Metastable RNA Structure

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

Hypothesis: type I toxin–antitoxin genes enter the persistence field—a feedback mechanism explaining membrane homoeostasis

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