Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis.

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.     

Real time kinetic studies of the interaction between folded antisense and target RNAs using surface plasmon resonance

Antisense RNAs interact with their complementary target RNAs as folded structures. The formation of early binding intermediates is the most important step in determining the overall rates of stable complex formation in vitro and the efficiency of control in vivo. In the case of CopA and CopT (antisense/target RNA pair of plasmid R1), recent studies have identified a four-way junction structure as the major binding intermediate. Previously, the kinetics of antisense/target RNA interaction was studied by indirect methods. Here we have used surface plasmon resonance to follow the binding of CopI (a truncated variant of CopA) to CopT in real time. A protocol was developed that permitted the determination of association and dissociation rate constants for wild-type and mutant CopI-CopT pairs. The K(D)-values calculated from these rate constants were in good agreement with the results obtained by indirect methods. In comparison to earlier model studies of interactions between simple complementary nucleic acids, we observe a different temperature dependence for dissociation rate constants. This may be indicative of the complexity of the steps required for interacting folded RNAs; intramolecular structure competes with intermolecular helix progression during complex formation. The association rate constants were not significantly dependent on temperature. The analysis presented shows that the stability of a kissing complex is not the primary determinant of the rate of stable CopA/CopT complex formation.     

Translational control by antisense RNA in control of plasmid replication

Control of replication of plasmids involves two processes: measurement of the copy number of the plasmid and adjustment of the replication frequency accordingly. For both these processes IncFII plasmids use an antisense RNA (CopA RNA) that forms a duplex with the upstream region (CopT) of the mRNA of the rate-limiting RepA protein. The kinetics of duplex formation was measured in vitro for the wild type and for a cop mutant plasmid; the mutant showed a reduction in the second-order rate constant for the formation of the RNA duplex and a similar increase in copy number. Hence, the kinetics of duplex formation and the concentration of CopA RNA determines the copy number of the plasmid.     

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.     

Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA-CopT complex is characterized by a four-way junction structure and a side-by-side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop-loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.     

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA–CopT complexes suggests that, subsequent to loop–loop contact, helix propagation is prevented. Instead, a fully base paired loop–loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop–loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.     

Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.     

Mechanism of binding of the antisense and target RNAs involved in the regulation of IncB plasmid replication.

The replication frequency of the IncB miniplasmid pMU720 is dependent upon the expression of the repA gene. Binding of a small, highly structured, antisense RNA (RNA I) to its complementary target in the RepA mRNA (RNA II) inhibits repA expression and thus regulates replication. Analyses of binding of RNA I to RNA II indicated that the reaction consists of three major steps. The first step, initial kissing complex formation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II. The second step is facilitated by interior loop structures in the upper stems of RNA I and RNA II and involves intrastand melting and interstrand pairing of the upper stem regions to form an extended kissing complex. This complex was shown to be sufficient for inhibition of repA expression. The third step involves stabilization of the extended kissing complex by pairing between complementary single-stranded tail regions of RNA I and RNA II. Thus, the final product of RNA I-RNA II binding is not a full duplex between the two molecules.     

An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1.

Antisense RNAs in prokaryotic systems often inhibit translation of mRNAs. In some cases, this involves sequestration of Shine-Dalgarno (SD) sequences and start codons. In other cases, antisense/target RNA duplexes do not overlap these signals, but form upstream. We have performed toeprinting analyses on repA mRNA of plasmid R1, both free and in duplex with the antisense RNA, CopA. An intermolecular RNA duplex 2 nt upstream of the tap SD prevents ribosome binding. An intrastrand stem-loop at this location yields the same inhibition. Thus, stable secondary structures immediately upstream of the tap SD sequence inhibit translation, as shown by toeprinting in vitro and repA-lacZ expression in vivo. Previous work showed that repA (initiator protein) expression requires tap (leader peptide) translation. Toeprinting data confirm that the tap ribosome binding site (RBS) is accessible, whereas the repA RBS, which is sequestered by a stable stem-loop, is weakly recognized by the ribosome. Truncated CopA RNA (CopI) is unable to pair completely with target RNA, but proceeds normally to a kissing intermediate. This mutant RNA species inhibits repA expression in vivo. By a kinetic toeprint inhibition protocol, we have shown that the structure of the kissing complex is sufficient to sterically prevent ribosome binding. These results are discussed in comparison with the effect of RNA structures elsewhere in the ribosome-binding region of an mRNA.     

Bulged-out nucleotides protect an antisense RNA from RNase III cleavage.

Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem-loop of the antisense RNA, CopA, carry bulged-out nucleotides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced. The set of mutant and wild-type CopA variants was characterized structurally in vitro. The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. In vitro cleavage rates were drastically increased when either the lower or both bulges were absent. This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo. The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed. In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo.     

Loop swapping in an antisense RNA/target RNA pair changes directionality of helix progression

The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.     

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli  ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.     

High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands

We have isolated 2′-Fluoro-substituted RNA aptamers that bind to streptavidin (SA) with an affinity around 7 ± 1.8 nM, comparable with that of recently described peptide aptamers. Binding to SA was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes. Mutagenesis, secondary structure analysis, ribonuclease footprinting and deletion analysis provided evidence for the essential structural features of SA-binding aptamers. In order to provide a general method for the exploitation of these aptamers, we produced derivatives in which they were fused to the naturally structured RNA elements, CopT or CopA. In parallel, we produced derivatives of CD4-binding aptamers fused to the complementary CopA or CopT elements. When mixed, these two chimeric aptamers rapidly hybridized, by virtue of CopA–CopT complementarity, to form stable, bi-functional aptamers that we called ‘adaptamers’. We show that a CD4–SA-binding adaptamer can be used to capture CD4 onto a SA-derivatized surface, illustrating their general utility as indirect affinity ligands.