Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
Microbiology (Reading). 2010 Dec;156(Pt 12):3609-3623. doi: 10.1099/mic.0.043745-0. Epub 2010 Aug 26.
pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (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 repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB 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 C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.
pCGR2 和 pCG1 属于谷氨酸棒杆菌 pCG1 家族质粒的不同亚家族。然而，它们分别含有同源的假定反义 RNA 基因 crrI 和 cgrI。这些基因反过来又具有与其各自 repA（编码质粒复制起始子）基因的先导区互补的相同位置。确定它们的确切转录起始和终止点揭示了短反义 RNA 分子（72 bp，CrrI；和 73 bp，CgrI）的存在。这些短 RNA 及其靶 mRNA 被预测形成包含已知 U 形转弯基序的茎环的高度结构化分子。通过启动子诱变消除 CrrI 和 CgrI 的合成导致质粒拷贝数增加了约七倍，而 repA mRNA 增加了 11 倍（CrrI）和 32 倍（CgrI），表明 CrrI 和 CgrI 负调控质粒复制。这种控制因 parB 而加剧，parB 是编码小的着丝粒结合质粒分配蛋白的基因，位于 repA 上游。CrrI 和 parB 的同时失活导致源自 pCGR2 的穿梭载体的拷贝数急剧增加 87 倍。此外，CrrI 和 CgrI 的末端环结构的变化影响质粒拷贝数的事实支持了环结构在反义 RNA 与其靶 mRNA 之间形成初始相互作用复合物中的重要作用。类似的反义 RNA 控制系统不仅可能存在于两个谷氨酸棒杆菌 pCG1 亚家族中，而且可能存在于相关的棒杆菌属质粒中。
