Control of Cell Division by Sex Factor F in Escherichia coli: Trigger protein and inhibitor protein encoded by the F plasmid.

大肠杆菌F质粒含有letA和letD两个基因，其产物参与了F质粒DNA复制与宿主细菌细胞分裂的偶联。letD基因产物抑制宿主细菌的细胞分裂，而letA基因产物抑制letD基因产物的抑制活性。当F质粒的DNA复制被阻断时，letA基因的表达被抑制，结果细胞停止分裂。为了阐明letA基因功能表达与F质粒DNA复制耦合的机制，我们构建了在letA启动子下游含有无启动子lacZ基因的mini-F质粒，并测量了-半乳糖苷酶活性。结果表明，letA和letD基因包含一个操纵子，该操纵子的表达在转录水平上受letA和letD基因产物的负调控，而在转录和翻译水平上不受F质粒DNA复制的负调控。此外，我们发现，即使该操纵子的启动子被tac启动子取代，F质粒的DNA复制与宿主细菌的细胞分裂之间的偶联仍被保留。这些观察结果表明，letA功能的表达在翻译后水平上受到DNA复制的调节。作为分析这种翻译后调控机制的第一步，我们试图寻找LetA和LetD蛋白携带的生化活性。通过凝胶阻滞分析，我们发现LetA蛋白与双链DNA的结合与碱基序列无关，同时我们也获得了LetA和LetD蛋白作为复合物与LetA、LetD操纵子的启动子区结合的证据，而宿主染色体编码的一种蛋白在这种结合中起着不可或缺的作用。利用这些特征来测定它们的活性，LetA和LetD蛋白的纯化正在进行中。

The F plasmid of Escherichia coli contains two genes, letA and letD, whose products are involved in the coupling between DNA replication of the F plasmid and cell division of the host bacteria. The letD gene product acts to inhibit cell division of the host bacteria, whereas the letA gene product acts to suppress the inhibitory activity of the letD gene product. When DNA replication of the F plasmid is blocked, expression of the letA gene is inhibited and as a result cells stop dividing.To elucidate the mechanism that couples expression of the letA gene function with DNA replication of the F plasmid, we constructed mini-F plasmids containing promoterless lacZ gene downstream of the letA promoter and measured -galactosidase activities. The results showed that the letA and letD genes comprise an operon, and that expression of this operon is regulated negatively at the level of transcription by the letA and letD gene products, but not by DNA replication of the F plasmid either at the level of transcription or translation. In addition, we showed that the coupling between DNA replication of the F plasmid and cell division of the host bacteria is preserved even if promoter of this operon was replaced with the tac promoter. These observations suggest that expression of the letA function is regulated by DNA replication at a post-translational level.As a first step to analyze this post-translational regulatory mechanism, we tried to search biochemical activities carried by LetA and LetD proteins. Using gel retardation analysis, we found that LetA protein binds to double stranded DNA indifferently to base sequence, and also obtained evidence that suggests LetA and LetD proteins, as a complex, binds to the promoter region of the letA, letD operon and that a protein encoded by the host chromosome plays indispensable role in this binding. Utilizing these features for assay of their activity, purification of LetA and LetD proteins is now under way.