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.     

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.     

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.     

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.     

Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.     

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.     

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid.

Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA. Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I [SLI]) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different. The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI. Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules. These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.     

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.     

Expression and regulation of the RepA protein of the RepFIB replicon from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene. Limited N-terminal sequence information obtained from a RepA:beta-galactosidase fusion protein indicates that although the first residue of RepA is methionine, the initiation of translation of RepA occurs from a CTG codon rather than from the predicted GTG codon located further downstream. Overexpressed RepA in trans is capable of repressing a repA:lacZ fusion plasmid in which the expression of the fusion protein is under the control of the repA promoter. The repA promoter has been located functionally by testing a series of repA:lacZ fusion plasmids. Both in vivo genetic tests and in vitro DNA-binding studies indicate that repA autoregulation can be achieved by RepA binding to one or more repeat elements which overlap the repA promoter sequence.     

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.     

Mutations affecting pseudoknot control of the replication of B group plasmids.

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.