Fujita M, Sadaie Y
Radioisotope Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
Gene. 1998 Oct 23;221(2):185-90. doi: 10.1016/s0378-1119(98)00452-1.
A highly ordered program of temporal and spatial gene activation during sporulation in Bacillus subtilis is governed by the principal RNA polymerase, and RNA polymerases containing at least five developmental sigma factors appearing successively during sporulation. This report describes a rapid procedure for extracting RNA polymerase from sporulating B. subtilis cells, which involves the construction of hexahistidine tagged beta' subunit of RNA polymerase and the isolation of RNA polymerase holoenzyme with Ni2+-NTA resin. In in vitro transcription of various promoters with the RNA polymerase thus purified, we observed the temporal change of each RNA polymerase activity during sporulation. This procedure enables isolation of RNA polymerase within 4h, starting with cell pellets. Our results indicated that a principal sigma factor, sigmaA, could be detected in a holoenzyme form during all the stages of growth and sporulation, while the other sigma factors sigmaH, sigmaE, sigmaF, sigmaG, and sigmaK involved in sporulation could be detected sequentially during sporulation. Moreover, Spo0A, the central transcription factor of commitment to sporulation, was also co-purified with RNA polymerase at early stages of sporulation.
枯草芽孢杆菌孢子形成过程中高度有序的时空基因激活程序由主要的RNA聚合酶以及在孢子形成过程中相继出现的至少五种发育型σ因子的RNA聚合酶控制。本报告描述了一种从孢子形成的枯草芽孢杆菌细胞中提取RNA聚合酶的快速方法，该方法涉及构建带有六聚组氨酸标签的RNA聚合酶β'亚基，并使用Ni2+-NTA树脂分离RNA聚合酶全酶。在用如此纯化的RNA聚合酶对各种启动子进行体外转录时，我们观察到了孢子形成过程中每种RNA聚合酶活性的时间变化。此方法能够从细胞沉淀开始，在4小时内分离出RNA聚合酶。我们的结果表明，主要的σ因子σA在生长和孢子形成的所有阶段都能以全酶形式被检测到，而参与孢子形成的其他σ因子σH、σE、σF、σG和σK在孢子形成过程中可依次被检测到。此外，孢子形成关键转录因子Spo0A在孢子形成早期也与RNA聚合酶共同纯化出来。
