Bowater R P, Chen D, Lilley D M
Department of Biochemistry, The University, Dundee, UK.
EMBO J. 1994 Dec 1;13(23):5647-55. doi: 10.1002/j.1460-2075.1994.tb06903.x.
We have found that initiation of RNA synthesis at the tyrT promoter of Escherichia coli can be stimulated on a plasmid by a factor of 4-6 by elevation of DNA supercoiling in vivo. Increased unconstrained plasmid supercoiling was achieved by inserting the tyrT promoter upstream of the tetracycline resistance gene tetA and transformation into a topA host. Under these conditions there is marked oversupercoiling of the plasmid DNA and we have shown previously that this can lead to increased promoter activity in the topological domain created. A critical element in the formation of this domain is the coupled transcription, translation and membrane insertion of tetA and we show that all of these events are important in the stimulation of tyrT promoter activity. The magnitude of the stimulation is in reasonable agreement with that measured in vitro as a function of plasmid supercoiling, if the unconstrained level of negative supercoiling in vivo is increased from a basal level of -sigma approximately 0.022 to -sigma approximately -0.052 by transcription-induced supercoiling. The induced supercoiling is very efficient, indicating that the tyrT promoter is itself contributing to the steady-state level despite a total lack of membrane anchorage for the tyrT transcription unit. This study provides a new example of the topological coupling of promoters.
我们发现，通过在体内提高DNA超螺旋程度，大肠杆菌tyrT启动子处的RNA合成起始在质粒上可被刺激4至6倍。通过将tyrT启动子插入四环素抗性基因tetA的上游并转化到topA宿主中，实现了质粒超螺旋的增加。在这些条件下，质粒DNA存在明显的过度超螺旋，并且我们之前已经表明，这会导致在形成的拓扑结构域中启动子活性增加。该结构域形成中的一个关键因素是tetA的转录、翻译和膜插入的偶联，并且我们表明所有这些事件在刺激tyrT启动子活性方面都很重要。如果通过转录诱导的超螺旋使体内负超螺旋的无约束水平从基础水平-σ约0.022增加到-σ约-0.052，那么刺激的幅度与体外作为质粒超螺旋函数测量的幅度合理一致。诱导的超螺旋非常有效，这表明尽管tyrT转录单元完全缺乏膜锚定，但tyrT启动子本身对稳态水平有贡献。这项研究提供了启动子拓扑偶联的一个新例子。
