The anti-σ factor SpoIIAB forms a 2:1 complex with σF, contacting multiple conserved regions of the σ factor
The anti-σ factor SpoIIAB forms a 2:1 complex with σF, contacting multiple conserved regions of the σ factor
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DOI：
10.1006/jmbi.2000.3838
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发表时间：
2000-06-30
影响因子：
5.6
通讯作者：
Darst, SA
中科院分区：
文献类型：
--
作者：
Campbell, EA;Darst, SA
枯草芽孢杆菌的发育调节蛋白sigma（F）是细菌RNA聚合酶sigma因子的sigma（70）-家族的成员，其被抗sigma因子SpoIIAB负调节，所述抗sigma因子SpoIIAB结合sigma（F），将其隔离在无活性复合物中。SpoIIAB与σ（F）的结合受到ATP的强烈刺激。在这里，我们使用凝胶过滤色谱，动态光散射，分析超离心，有限的蛋白水解与N-末端测序和电喷雾质谱，和缺失分析的组合来探测SpoIIAB-sigma（F）复合物。通过研究嗜热脂肪芽孢杆菌的同源物以及在大肠杆菌中共表达蛋白质，从而纯化大量体内组装的复合物，促进了研究。我们确定络合物的化学计量为SpoIIAB（2）：sigma（1）（F）。单独的sigma（F）被蛋白酶胰蛋白酶迅速降解。然而，在与SpoIIAB的复合物中，sigma（F）对蛋白水解具有显著的抗性。对蛋白酶切割数据的分析表明，反σ通过与σ因子的多个保守区域接触而结合σ（F），支持了基于遗传数据的先前发现。(C)北京大学出版社.
The developmental regulatory protein sigma(F) of Bacillus subtilis, a member of the sigma(70)-family of bacterial RNA polymerase sigma factors, is negatively reguL lated by the anti-sigma factor SpoIIAB, which binds to sigma(F), sequestering it in an inactive complex. SpoIIAB binding to sigma(F) is strongly stimulated by ATP. Here, we use a combination of gel filtration chromatography, dynamic light-scattering, analytical ultracentrifugation, limited proteolysis with N-terminal sequencing and electrosyray mass spectrometry, and deletion analysis to probe the SpoIIAB-sigma(F) complex. The studies were facilitated by investigating the homologs from Bacillus stearothermophilus as well as co-expression of the proteins in Escherichia coli, allowing purification of large quantities of the in vivo assembled complex. We determined the stoichiometry of the complex to be SpoIIAB(2):sigma(1)(F). Alone, sigma(F) is rapidly degraded by the protease trypsin. In the complex with SpoIIAB, however, sigma(F) is remarkably resistant to proteolysis. Analysis of the protease cleavage data indicates the anti-sigma binds sigma(F) through contacts with multiple conserved regions of the sigma factor, supporting previous findings based on genetic data. (C) 2000 Academic Press.