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Volume 156, Issue 12

Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Free

Abstract

pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, and , respectively. The genes in turn share identical positions complementary to the leader region of their respective (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem–loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of . Simultaneous deactivation of CrrI and led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two pCG1 subfamilies but also in related plasmids across species.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): RACE, rapid amplification of cDNA ends , RT-qPCR, real-time quantitative RT-PCR and SD, Shine–Dalgarno
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2010-12-01
2024-04-19
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Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum
Microbiology 156, 3609 (2010); https://doi.org/10.1099/mic.0.043745-0
/content/journal/micro/10.1099/mic.0.043745-0
/content/journal/micro/10.1099/mic.0.043745-0
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