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.     

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.     

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.     

Copy mutants of plasmid R1: effects of base pair substitutions in the copA gene on the replication control system

Summary Five different copA copy mutants of plasmid R1 have been identified by nucleotide sequencing. Independent measurements of the activities of the mutant inhibitor RNA and of the mutant target properties were carried out using several different methods. Correlation of these measurements with the location of the nucleotide substitutions resulted in the following conclusions: (1) The copy number of plasmid R1 is controlled primarily by interaction between the CopA RNA molecule and its target, the RepA mRNA. (2) The binding of the inhibitor to its target is based on nucleotide interactions within two complementary sequences of ten nucleotides and dependent on the secondary structure of the active site. (3) The secondary structure of both the CopA target and the CopA RNA is a stem-loop structure. Mutations in the loop region interfere with binding affinity between inhibitor and target, whereas mutations in the upper stem mainly interfere with secondary structure. Mutations in the latter region create temperature-dependent copy number phenotypes.     

Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo.

Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.     

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.     

Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA--target RNA pairing.

The replication frequency of plasmid R1 is determined by the availability of the initiator protein RepA. Synthesis of RepA is negatively controlled by an antisense RNA, CopA, which forms a duplex with the upstream region of the RepA mRNA, CopT. We have previously shown that the in vitro formation of the CopA-CopT duplex follows second-order kinetics and occurs in at least two steps. The first step is the formation of a transient (kissing) complex, which is subsequently converted to a persistent duplex. Here, we investigate the details of the reaction scheme and determine the rate constants of the pathway from the free RNAs to the complete duplex. Using a shortened CopA RNA (CopI) we have been able to determine the association and dissociation rate constants (k1,k-1) for the kissing complex (which are inferred to be the same for CopI-T and CopA-T), and measured the hybridization rate constant k2 (for CopA-T k2 is at least 1000-fold greater than for CopI-T). The analysis of CopA derivatives of mutant and wild-type origin shows that the rate of formation of the kissing complex is rate-limiting for the overall pairing reaction between CopA and CopT, both in vitro and in vivo. The biological implications of the kinetically irreversible RNA-RNA binding reaction scheme 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.     

Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. In plasmid R1, we have recently shown that the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by the formation of two intermolecular helices, resulting in a four-way junction structure and a side-by-side helical alignment. Based on lead-induced cleavage and ribonuclease (RNase) V(1) probing combined with molecular modeling, a strikingly similar topology is supported for the complex formed between the antisense RNA (Inc) and mRNA (RepZ) of plasmid Col1b-P9. In particular, the position of the four-way junction and the location of divalent ion-binding site(s) indicate that the structural features of these two complexes are essentially the same in spite of sequence differences. Comparisons of several target and antisense RNAs in other plasmids further indicate that similar binding pathways are used to form the inhibitory antisense-target RNA complexes. Thus, in all these systems, the structural features of both antisense and target RNAs determine the topologically possible and kinetically favored pathway that is essential for efficient in vivo control.     

Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral RNA replication

The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.     

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.