Control of replication of plasmid R1: kinetics of in vitro interaction between the antisense RNA, CopA, and its target, CopT.

Persson, C.; Wagner, E. Gerhart H.; Nordström, Kurt

doi:10.1002/j.1460-2075.1988.tb03195.x

Cited by 117 publications

(162 citation statements)

References 45 publications

(28 reference statements)

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“…In naturally occurring antisense RNA-regulated systems, RNA secondary structures are known to influence the kinetics of double strand formation in vitro [35]. These measured differences are directly correlated with biological effectiveness in vivo [36][37][38][39]. A similar correlation between RNA-RNA annealing kinetics in vitro and efficacy in human cells has been observed for artificial Antisense RNAs directed against HIV-1 [40,41].…”

Section: Discussionmentioning

confidence: 70%

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

Koslowsky¹,

Reifur²,

Yu³

et al. 2004

RNA Biology

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The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K D in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

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Section: Discussionmentioning

confidence: 70%

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

Koslowsky¹,

Reifur²,

Yu³

et al. 2004

RNA Biology

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“…Interaction of such highly structured antisense RNA molecules with their targets is likely to involve several steps and starts with the formation of so-called kissing complexes between the singlestranded loops of antisense and target RNA. This concept was initially proposed by for the interaction of RNAI and RNAII of ColEl and was later supported by both in vivo and in vitro studies of the interaction of the CopA antisense RNA of plasmid R1 with its target CopT (31)(32)(33). In vitro experiments with the R1 system even suggested that formation of the kissing complex is sufficient for the inhibitory action of CopA to occur (43).…”

Section: Materlils and Methodsmentioning

confidence: 89%

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

Brantl¹,

Birch-Hirschfeld²,

Behnke³

1993

J Bacteriol

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Expression of the rate-limiting initiator protein RepR of plasmid pIP501 is controlled by the antisense RNAIII. Mutational alteration of individual G residues within the single-stranded loops of RNAIII led to an increase in copy number. In contrast to the G-rich single-stranded loops, two smaller AT-rich loops of RNAIII were found to be dispensable for its inhibitory function. Reciprocal mutations in the same loop compensated for each other's effect, and a destabilization of the major stem structure of RNAIII also resulted in an increased copy number. These data were consistent with the idea that the interaction of RNAIII with its target starts with the formation of a kissing complex between the single-stranded loops of both molecules. The repR mRNA leader sequence, which includes the target of RNAIIIs is able to assume two alternative structures due to the presence of two inverted repeats the individual sequences of which are mutually complementary. In the presence of the antisense RNAIII, one of these inverted repeats (IR2) is forced to fold into a transcriptional terminator structure that prevents transcription of the repR gene. In the absence of RNAIII, formation of the transcriptional terminator is prevented and expression of the essential repR gene can proceed normally. This antisense RNA-driven transcriptional attenuation mechanism was supported by extensive deletional analysis and direct evidence that IR2 functions as a transcriptional terminator.

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“…Sok RNA and variants of truncated hok mRNA were synthesized in vitro, gelpurified, and renatured. Using an in vitro binding assay (38), the apparent secondorder binding-rate constants (K app ) of Sok RNA association with truncated wild-type and mutant hok mRNAs were determined (Table I). Sok RNA bound with similar high rates to truncated wild-type and super-tac hok mRNAs.…”

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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Control of replication of plasmid R1: kinetics of in vitro interaction between the antisense RNA, CopA, and its target, CopT.

Cited by 117 publications

References 45 publications

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

Temporal Translational Control by a Metastable RNA Structure

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