Thisted T, Sørensen N S, Gerdes K
Department of Molecular Biology, Odense University, Denmark.
J Mol Biol. 1995 Apr 14;247(5):859-73. doi: 10.1006/jmbi.1995.0186.
The hok/sok system of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation of hok is coupled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translation indirectly through mok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was active. We have therefore previously suggested, that the 3'-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termed fbi (fold-back inhibition). Here we describe the in vitro secondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of mok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3'-end fbi element. These "structure closing" mutations affected the structure much further upstream in the mok translational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.
质粒R1的hok/sok系统通过杀死无质粒细胞来介导质粒稳定化。Hok mRNA非常稳定，能够被翻译成Hok杀伤蛋白。Hok mRNA的翻译受到小的不稳定的Sok反义RNA的抑制。hok的翻译与一个称为mok的重叠阅读框偶联。mok的翻译受到Sok RNA的严格调控，因此Sok RNA通过mok间接调控hok的翻译。Sok RNA的快速降解解释了新生无质粒分离体中Hok合成的起始。然而，在这个简单的诱导机制之上叠加了第二个控制水平，因为发现全长Hok 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Hok mRNA的3'末端区域编码一个阻止其翻译的元件。这个元件被称为fbi（回折抑制）。在这里，我们描述了整个Hok mRNA的体外二级结构。我们的结果表明存在一种封闭结构，其中全长Hok mRNA的3'端回折到mok的翻译起始区域。这种结构解释了为什么全长Hok mRNA没有翻译活性。使用3'端fbi元件中的突变获得的结果进一步支持了所提出的结构。这些“结构封闭”突变在mok翻译起始区域更上游的位置对结构产生了更大影响，并同时阻止了反义RNA与mRNA的同一区域结合。这些结果进一步支持了解释无质粒分离体中Hok mRNA翻译起始的诱导模型。该模型中最重要的调控元件是在3'端和mok翻译起始区域之间形成的FBI结构。这种结构使Hok mRNA没有翻译活性并阻止反义RNA结合，从而允许积累一批mRNA，这些mRNA通过缓慢的3'端加工在无质粒分离体中被激活，最终导致这些细胞被清除。
