Nielsen A K, Gerdes K
Department of Molecular Biology, Odense University, Denmark.
J Mol Biol. 1995 Jun 2;249(2):270-82. doi: 10.1006/jmbi.1995.0296.
The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells. The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane. Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt. The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin. Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active. We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation. Here we present an analysis of the metabolism of the pnd mRNAs. A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD. Mutations in the anti-SD element were lethal. These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element). These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA. This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin.
质粒R483的pnd系统通过杀死无质粒细胞来介导质粒稳定。pnd mRNA非常稳定，可被翻译成PndA蛋白，一种细胞毒素，它通过破坏细胞膜从内部杀死细胞。pnd mRNA的翻译受到PndB反义RNA的抑制，PndB反义RNA是一种63个核苷酸的不稳定小RNA。PndB解毒剂的快速降解导致在无质粒分离子中或添加利福平后开始合成PndA。然而，令人惊讶的是，发现全长pnd mRNA没有翻译活性，而其3'端截短版本具有活性。因此，我们之前提出，全长pnd mRNA的3'端编码一个回折抑制序列（fbi），它阻止其翻译。在此，我们对pnd mRNA的代谢进行分析。突变分析表明，fbi基序中的单点突变导致更快的截短。fbi突变不能被pndA或pndC的Shine-Dalgarno（SD）元件中的第二位点突变互补。令人惊讶的是，pndC SD元件中的突变也导致更快的截短。然而，后一种突变的影响被pndC SD上游一个假定的反SD元件中的突变互补。反SD元件中的突变是致死的。这些结果表明，pnd mRNA包含两个负调控元件，一个位于其3'端（fbi），另一个位于pndC SD区域上游（反SD元件）。这些观察结果增加了先前提出的解释无质粒细胞中pndA表达激活的诱导方案的复杂性：除了对翻译的负面影响外，fbi结构还维持mRNA 3'端较低的加工速率。这允许积累pnd mRNA库，其可在无质粒细胞中通过3'端加工被激活。反SD可能在转录过程中阻止pnd mRNA的翻译，从而防止毒素的有害合成。
