Koraimann G, Teferle K, Markolin G, Woger W, Högenauer G
Institut für Mikrobiologie, Karl-Franzens-Universität Graz, Austria.
Mol Microbiol. 1996 Aug;21(4):811-21. doi: 10.1046/j.1365-2958.1996.361401.x.
A key determinant of the frequency of IncF plasmid-mediated DNA transfer between enterobacterial cells is the FinOP system. traJ, a positive regulator of the transfer (tra) genes is controlled at the post-transcriptional level by two negative elements, finP and finO. FinP is a plasmid-specific antisense RNA, whereas finO encodes a proteic co-repressor which is not plasmid specific but exchangeable among F-like plasmids. We designed a traJ-lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression. Furthermore, the introduction of finO into the test system enabled us to assess the function of FinO in the interaction of FinP with its target, the traJ mRNA. In this test system, FinP, expressed from a single-copy plasmid, in the absence of FinO, repressed traJ expression six-fold. When expressed from a pBR322-derived multicopy plasmid FinP repressed traJ expression approx. 2000-fold. This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage-dependent manner. Mutations of finP creating base exchanges either in loop I or loop II of the two stem-loop structures of the antisense RNA led to a dramatic decrease in the repressor activity. In a combined loop I-loop II mutation the repressor activity was almost completely lost, supporting the model that the first critical interaction between the two RNA molecules occurs via 'kissing' of both loops of the RNAs. Addition of finO to the test system enhanced the repression of traJ expression by FinP by up to two orders of magnitude. This effect of FinO on FinP activity in vivo might indicate that FinO, in addition to its function as an RNA stabilizer, promotes complex formation between the target mRNA and the antisense RNA. Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbiol 14: 427-436).
IncF 质粒介导的肠杆菌细胞间 DNA 转移频率的一个关键决定因素是 FinOP 系统。traJ 是转移（tra）基因的正调控因子，在转录后水平受两个负调控元件 finP 和 finO 的控制。FinP 是一种质粒特异性反义 RNA，而 finO 编码一种蛋白质共抑制因子，它不是质粒特异性的，但可在 F 样质粒之间交换。我们设计了一个 traJ-lacZ 测试系统，使我们能够监测 FinP 和各种 FinP 突变体对 traJ 表达的影响。此外，将 finO 引入测试系统使我们能够评估 FinO 在 FinP 与其靶标 traJ mRNA 相互作用中的功能。在这个测试系统中，从单拷贝质粒表达的 FinP，在没有 FinO 的情况下，使 traJ 表达受到六倍的抑制。当从 pBR322 衍生的多拷贝质粒表达时，FinP 使 traJ 表达受到约 2000 倍的抑制。这一结果明确表明，FinP 足以以基因剂量依赖的方式抑制 traJ 表达。在反义 RNA 的两个茎环结构的环 I 或环 II 中产生碱基交换的 finP 突变导致阻遏活性显著降低。在环 I - 环 II 组合突变中，阻遏活性几乎完全丧失，支持了两个 RNA 分子之间的首次关键相互作用通过 RNA 两个环的“亲吻”发生的模型。向测试系统中添加 finO 可使 FinP 对 traJ 表达的抑制作用增强多达两个数量级。FinO 在体内对 FinP 活性的这种作用可能表明，FinO 除了作为 RNA 稳定剂的功能外，还促进靶标 mRNA 与反义 RNA 之间的复合物形成。最近已在体外证明 FinO 具有这种功能（van Biesen 和 Frost（1994 年），《分子微生物学》14：427 - 436）。
