Plasmid Replication Control by Antisense RNAs

FIGURE 1

Mechanisms of antisense-RNA-mediated plasmid copy number control. Antisense RNAs are drawn in red; sense RNAs are drawn in blue. ORFs encoding essential replication initiator proteins are shown as orange boxes; ORFs encoding transcriptional repressor proteins are shown as brown boxes. SD sequences for rep ORFs are blue rectangles. Promoters are symbolized by black triangles and replication origins by dark grey ovals. Arrows indicate positive interaction; black bars indicate repression. Ribosomes are in light yellow. (A) Transcriptional attenuation: plasmid pIP501. (Upper part) Working model on regulation of pIP501 replication. The minimal replicon with the copR and repR genes is shown, separated by the 329-nt-long leader region. CopR represses transcription from the repR promoter pII and, at the same time, indirectly increases transcription initiation from the antisense promoter pIII. The antisense RNA causes premature termination of repR (sense) RNA transcription at the attenuator (att). (Lower part) Mechanism of transcriptional attenuation. For details see text. Complementary sequence elements are designated A, B, a, and b. Green arrow, RNase III. (B) Inhibition of primer maturation: plasmid ColE1. (Upper part) Schematic representation of the minimal replicon. (Lower part) Mechanism of inhibition of primer maturation. Violet circle, RNA polymerase. For details, see text. (C) Inhibition of pseudoknot formation: plasmid ColIb-P9. (Upper part) The minimal replicon with the leader peptide repY (dark grey) and repZ genes is shown. White: leader region of repZ mRNA. (Lower part) Genes for repY and repZ are translationally coupled. On the mRNA, the repY SD sequence is exposed, whereas structure III sequesters both the repZ SD sequence (black rectangle) and the 5′-rCGCC-3′ sequence (thick black line) and, thereby, repZ translation. Inc is the region complementary to the antisense RNA; black circle, repY start codon; grey circle, repY stop codon. Unfolding of structure II by the ribosome stalling at the repY stop codon results in formation of a pseudoknot by base paring between the 5′-rGGCCG-3′ and 5′-CGCC-3′ (thick black lines) sequences distantly separated, and allows the ribosome to access the repZ RBS. Binding of Inc RNA to the loop of structure I of repZ RNA directly inhibits formation of the pseudoknot and the subsequent IncRNA-repZ-mRNA duplex formation inhibits repY translation. (D) Translation inhibition by inhibition of ribosome binding. (Upper part) Working model on regulation of plasmid pMV158 replication. (Lower part) The antisense RNA binds directly upstream of the extended non-SD sequence (light blue circle) 5′ of the repB start codon and prevents binding of the 30S ribosomal subunit. The CopG protein represses transcription from the copG-repB-promoter and from the repB promoter. (E) Inhibition of leader peptide translation. (Upper part) Working model on regulation of plasmid R1 replication. (Lower part) Translation of the leader peptide (black box) tap is required for efficient repA translation. The CopB protein represses transcription from the repA, but not from the copB promoter. (F) Translational attenuation. (Upper part) Working model on regulation of plasmid pSK41 replication. (Lower part) The antisense RNA interacts via three loops with the nascent repA mRNA resulting in a stem-loop structure that sequesters the ribosome binding site. In the absence of RNAI, the repA mRNA refolds into an alternative structure that exposes the ribosome binding site, allowing repA translation. .