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Antisense and yet sensitive: Copy number control of rolling circle‐replicating plasmids by small RNAs

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Bacterial plasmids constitute a wealth of shared DNA amounting to about 20% of the total prokaryotic pangenome. Plasmids replicate autonomously and control their replication by maintaining a fairly constant number of copies within a given host. Plasmids should acquire a good fitness to their hosts so that they do not constitute a genetic load. Here we review some basic concepts in plasmid biology, pertaining to the control of replication and distribution of plasmid copies among daughter cells. A particular class of plasmids is constituted by those that replicate by the rolling circle mode (rolling circle‐replicating [RCR]‐plasmids). They are small double‐stranded DNA molecules, with a rather high number of copies in the original host. RCR‐plasmids control their replication by means of a small short‐lived antisense RNA, alone or in combination with a plasmid‐encoded transcriptional repressor protein. Two plasmid prototypes have been studied in depth, namely the staphylococcal plasmid pT181 and the streptococcal plasmid pMV158, each corresponding to the two types of replication control circuits, respectively. We further discuss possible applications of the plasmid‐encoded antisense RNAs and address some future directions that, in our opinion, should be pursued in the study of these small molecules. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
Regulatory circuit of plasmid pMV158. Nucleotide positions with reference to the adenine of the repB AUG start codon are indicated. (a) Diagrammatic representation of the secondary structures of repB mRNA (cyan), counter‐transcript RNAII (red) and their complex (cyan‐red; with // indicating a part of the duplex that is not shown). Thin lines in repB mRNA structure indicate alternative pseudoknots. Upon mRNA/RNAII duplex formation and structural switch of repB mRNA, translating ribosomes would not be able to bind to repB translation initiation site anymore. (b) Sequence and secondary structure of repB mRNA. In bold are shown bases with positions conserved at least with 75% (using an extended nucleotide “alphabet,” where R = A/G and W=U/A; alignment from (López‐Aguilar et al., ). Promoter PctII (‐35 and ‐10 sequences indicated) directs synthesis of RNAII that starts 17 bases before the start codon of repB (highlighted in green) and terminates past the UAA stop codon of copG (highlighted in red). Red bases indicate ARBS, and bases highlighted in yellow show wSD (GGGU). Boxed regions highlight mRNA and RNAII mutants that show impaired function (López‐Aguilar et al., ; López‐Aguilar et al., ; López‐Aguilar & del Solar, ). The RNAII transcript (‐17 to ‐64) overlaps partially the ARBS/wSD sequences of gene repB. In the lower part of the Figure, a dot‐bracket notation of repB mRNA secondary structure (matching brackets symbolize base pairs and dots show unpaired bases) for mRNA alone (with three alternative pseudoknots) and mRNA upon pairing with RNAII are shown
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Regulatory circuit of plasmid pT181 indicated by a schematic representation of the interactions between the counter‐transcribed RNA (RNAI, red) and the repC mRNA (cyan). The early transcript (~110 nt‐long) of the repC mRNA would fold as having two main hairpins. In the absence of RNAI, the late transcript would pleat as a complex structure that would place the ribosome binding site and the repC initiation codon accessible to the translating ribosomes (upper part of the diagram). However, and within a time‐window, pairing between the complementary loops of the hairpins generated in the repC mRNA and RNAI molecules may take place. In this case, generation of a hybrid RNAI and mRNA would lead to profound conformational changes in the duplex RNA. In this case, the downstream region of the duplex would generate a new hairpin ending in a 5′‐UUUUUUAU‐3′ sequence with the configuration of a rho‐independent transcription terminator. The result would be an untranslated truncated RNA duplex (lower part)
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Regulatory circuits of RCR‐plasmids pT181 and pMV158. (a) In the case of pT181, promoter Prep directs synthesis of the RepC initiator (green ellipse), which initiates replication by binding to its cognate dso. Replication is controlled by RNAI (red) that is complementary to the untranslated 5′‐end of the repC mRNA. (b) For pMV158, the circuit is regulated by the replication initiator protein RepB (green ellipse) that initiates replication by binding to the dso and cleaving the phosphodiester bond of a specific dinucleotide, leaving a 3′‐OH end that acts as the replicative primer (see also Figure ). The negatively acting elements (red) are the transcriptional repressor protein CopG and the antisense RNAII. The mechanism for controlling synthesis of RepB is provided by both elements: CopG represses synthesis of the initiator by binding to the Pcr promoter, whereas binding of RNAII to the copGrepB mRNA leads to inhibition of the binding of the ribosomes in the mRNA
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Features of plasmid copy numbers in their bacterial hosts. (a) The theoretical distribution of plasmid copy numbers at the moment of cell division is provided by a Gaussian curve, in which the Nav is indicated by a vertical dotted line. Plasmids having a narrow distribution (red) will have more probabilities to exhibit a stable inheritance than plasmids with a broad distribution (black). (b) Rate of plasmid loss in cultures grown during n generations in the absence of selective pressure. Stable (red line) and unstable (black line) plasmid inheritance are depicted. (c) Copy number fluctuations in the Nav of a plasmid following colonization of a new bacterial host
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Three‐dimensional structural models of pMV158 copG‐repB mRNA intergenic region (cyan), RNAII (red), and their early and final complex (all models were submitted to RNArchitecture database (Boccaletto et al., ). The models were predicted by SimRNAweb (Magnus, Boniecki, Dawson, & Bujnicki, ) with secondary structure inputs as predicted by RNA structure (Bellaousov, Reuter, Seetin, & Mathews, ), and experimentally evaluated (fully for RNAII and partially for the mRNA intergenic region) by López‐Aguilar in G. del Solar laboratory (López‐Aguilar & del Solar, ; López‐Aguilar, Romero‐López, Espinosa, Berzal‐Herranz, & del Solar, ). Figures were generated using PyMol (DeLano, )
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Simplified model of plasmid replication by the rolling circle mechanism. The initiator of replication (Rep) dimeric protein (red ellipses) recognizes, binds to it, and cleaves the phosphodiester bond of a specific dinucleotide (within a stem‐loop structure, represented by a gray circle) at the origin of replication of the leading strand (dso) on supercoiled DNA. This reaction leaves a 3′‐OH end that will be used as primer to assemble the replisome. The various stages of the process (a–f) are detailed in the text. the single‐stranded origin of replication, sso (filled circle in blue), is generated in the ssDNA intermediate by intrastrand pairing. More detailed representations of the mechanism of replication by the RC‐mode have been published (Espinosa, ; Khan, ; Lorenzo‐Díaz, Fernández‐López, Garcillán‐Barcia, & Espinosa, ; Ruiz‐Masó et al., )
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RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs

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