Alper S, Dufour A, Garsin D A, Duncan L, Losick R
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
J Mol Biol. 1996 Jul 12;260(2):165-77. doi: 10.1006/jmbi.1996.0390.
The RNA polymerase sigma factor sigma B is a stress-response regulatory protein in Bacillus subtilis. The activity of sigma B is controlled in part by RsbW, a protein that inhibits sigma B, and RsbV, a protein that counteracts this inhibition. We now demonstrate that purified RsbW is capable of forming alternative complexes with either sigma B or RsbV. Sigma B in the RsbW. sigma B complex was transcriptionally inactive. RsbV reversed this inhibition by sequestering RsbW in a RsbW-RsbV complex, thereby allowing sigma B to remain free and active. In contrast to interactions among the components of the homologous regulatory system for the sporulation transcription factor sigma F, the binding of RsbW to RsbV and sigma B did not require adenosine nucleotides. Experiments involving the exchange of proteins between the two regulatory systems demonstrated that RsbW and its homolog in the sigma F system, SpoIIAB, exhibit strong preference in binding to RsbV and sigma B, and SpoIIAA and sigma F, respectively, and that the difference in nucleotide-dependence of binding between these two systems is attributable to a difference between RsbW and SpoIIAB. In confirmation and extension of previous results, we show that RsbW is also a protein kinase that uses ATP to phosphorylate RsbV, thereby blocking the capacity of RsbV to bind to RsbW and activate transcription. A close correlation was observed between the concentration of ATP required for efficient RsbW-mediated phosphorylation of RsbV, inhibition of RsbW.RsbV comlex formation, and inhibition of sigma B-directed transcription. These results are consistent with the hypothesis that activation of sigma B under certain stress condition is due to a decrease in cellular ATP levels.
RNA聚合酶σ因子σB是枯草芽孢杆菌中的一种应激反应调节蛋白。σB的活性部分受RsbW（一种抑制σB的蛋白）和RsbV（一种抵消这种抑制作用的蛋白）的控制。我们现在证明，纯化的RsbW能够与σB或RsbV形成不同的复合物。RsbW-σB复合物中的σB没有转录活性。RsbV通过将RsbW隔离在RsbW-RsbV复合物中来逆转这种抑制作用，从而使σB保持游离并具有活性。与芽孢形成转录因子σF的同源调节系统各组分之间的相互作用不同，RsbW与RsbV和σB的结合不需要腺苷核苷酸。涉及两个调节系统之间蛋白质交换的实验表明，RsbW及其在σF系统中的同源物SpoIIAB分别对与RsbV和σB以及SpoIIAA和σF的结合表现出强烈的偏好，并且这两个系统之间结合的核苷酸依赖性差异归因于RsbW和SpoIIAB之间的差异。为了证实和扩展先前的结果，我们表明RsbW也是一种蛋白激酶，它利用ATP将RsbV磷酸化，从而阻断RsbV与RsbW结合并激活转录的能力。在有效进行RsbW介导的RsbV磷酸化所需的ATP浓度、RsbW-RsbV复合物形成的抑制以及σB指导的转录抑制之间观察到密切的相关性。这些结果与以下假设一致：在某些应激条件下σB的激活是由于细胞ATP水平的降低。
