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.     

Antisense RNAs in plasmids: control of replication and maintenance

The search for small RNAs which might act as riboregulators became successful over the past two years both in prokaryotes and in eukaryotes. Moreover, artificially designed antisense RNAs have become powerful tools to downregulate the expression of targeted genes. It seems that antisense RNAs as regulatory molecules are most likely to be found everywhere. However, the first naturally occuring antisense RNAs were identified in plasmids and other prokaryotic accessory DNA elements. The thorough and detailed analyses of these systems have provided deep insights into structure and function of prokaryotic antisense RNAs and the kinetics of antisense/sense RNA interaction. Here, I focus on the role of antisense RNAs in plasmid replication and maintenance.     

U-turns and regulatory RNAs

Conventional antisense RNAs, such as those controlling plasmid replication and maintenance, inhibit the function of their target RNAs rapidly and efficiently. Novel findings show that a common U-turn loop structure mediates fast RNA pairing in the majority of these RNA controlled systems. Usually, an antisense RNA regulates a single, cognate target RNA only. Recent reports, however, show that antisense RNAs can act as promiscuous regulators that control multiple genes in concert to integrate complex physiological responses in Escherichia coli.     

Antisense RNA regulation by stable complex formation in the Enterococcus faecalis plasmid pAD1 par addiction system

The par stability determinant, encoded by the Enterococcus faecalis plasmid pAD1, is the only antisense RNA regulated postsegregational killing system identified in gram-positive bacteria. Because of the unique organization of the par locus, the par antisense RNA, RNA II, binds to its target, RNA I, at relatively small, interspersed regions of complementarity. The results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other antisense RNA regulated systems, RNA I and RNA II form a relatively stable, presumably translationally inactive complex. The stability of the RNA I-RNA II complex would allow RNA I to persist in an untranslated state unless or until the encoding plasmid was lost. After plasmid loss, RNA II would be removed from the complex, allowing translational activation of RNA I. The mechanism of RNA I activation in vivo is unknown, but in vitro dissociation experiments suggest that active removal of RNA II, for example by a cellular RNase, may be required.     

Structural analysis of late intermediate complex formed between plasmid ColIb-P9 Inc RNA and its target RNA. How does a single antisense RNA repress translation of two genes at different rates?

The antisense Inc RNA encoded by the IncIalpha ColIb-P9 plasmid replicon controls the translation of repZ encoding the replication initiator and its leader peptide repY at different rates with different mechanisms. The initial loop-loop base pairing between Inc RNA and the target in the repZ mRNA leader inhibits formation of a pseudoknot required for repZ translation. A subsequent base pairing at the 5' leader of Inc RNA blocks repY translation. To delineate the molecular basis for the differential control, we analyzed the intermediate complexes formed between RepZ mRNA and Inc RNA(54), a 5'-truncated Inc RNA derivative. We found that the initial base pairing at the loops transforms into a more stable intermediate complex by its propagation in both directions. The resulting extensive base pairing indicates that the inhibition of the pseudoknot formation is established at this stage. Furthermore, the region of extensive base pairing includes bases different in related plasmids showing different incompatibility. Thus, the observed extensive base pairing is important for determining the incompatibility of the low-copy-number plasmids. We discuss the evolution of replication control systems found in IncIalpha, IncB, and IncFII group plasmids.     

Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site.

Replication of a low-copy-number IncIalpha plasmid ColIb-P9 depends on expression of the repZ gene encoding the replication initiator protein. repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem-loop I, the Inc RNA target, and a downstream sequence complementary to the loop I. The loop I sequence comprises 5'-rUUGGCG-3', conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control. Here we show that the level of repZ expression is rate-limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb-P9 derivatives defective in Inc RNA and pseudoknot formation. Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I. This initial interaction is stimulated by the presence of the loop U residue in the 5'-rUUGGCG-3' motif. These results indicate that the competition between the two RNA-RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.     

Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis

RNA loop-loop complexes are motifs that regulate biological functions in both prokaryotic and eukaryotic organisms. In E. coli, RNAI, an antisense RNA encoded by the ColE1 plasmid, regulates the plasmid replication by recognizing through loop-loop interactions RNAII, the RNA primer that binds to the plasmidic DNA to initiate the replication. Rop, a plasmid-encoded homodimeric protein interacts with this transient RNAI-RNAII kissing complex. A surface plasmon resonance (SPR)-based biosensor was used to investigate this protein-nucleic acid ternary complex, at 5 degrees C, in experimental conditions such as the protein binds either to the loop-loop complex while it dissociates or to a saturated stable RNAI-RNAII surface. The results show that RNAI hairpin dissociates from the RNAII surface 110 times slower in the presence of Rop than in its absence. Rop binds to RNAI-RNAII with an on-rate of 3.6 x 10(6) M(-1) s(-1) and an off-rate of 0.11 s(-1), resulting in a binding equilibrium constant equal to 31 nM. A Scatchard-plot analysis of the interaction monitored by SPR confirms a 1:1 complex of Rop and RNAI-RNAII as observed for non-natural Rop-loop-loop complexes.     

Replication and Control of Circular Bacterial Plasmids

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.