Rowe-Magnus D A, Spiegelman G B
Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3.
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5305-10. doi: 10.1073/pnas.95.9.5305.
Spo0A is the central regulator of commitment to sporulation in Bacillus subtilis. Spo0A is a member of the response regulator family of proteins and both represses and stimulates transcription from promoters when activated. In vivo Spo0A activation takes place by phosphorylation and in vitro activation can be accomplished by phosphorylation or removal of the N-terminal domain of the protein. We have examined the mechanism of Spo0A stimulation of transcription from the promoter of the spoIIG operon. This operon encodes one of the first compartment specific sigma factors whose appearance regulates sporulation development. When activated Spo0A was incubated with RNA polymerase and a DNA fragment containing the spoIIG promoter, bases between -13 and -3, relative to the start site of transcription, were denatured. Addition of activated Spo0A or RNA polymerase alone did not induce denaturation. Heteroduplex templates that contained the nontemplate sequence of the wild-type promoter on both strands between positions -3 and -13 were efficiently transcribed without activated Spo0A. These data suggest that DNA strand separation is a two-step process and that the activation of Spo0A creates a form that interacts with the polymerase to induce the first of the two steps.
Spo0A是枯草芽孢杆菌中孢子形成过程的核心调控因子。Spo0A是应答调控蛋白家族的成员，激活后既可以抑制也可以刺激启动子的转录。在体内，Spo0A通过磷酸化激活，在体外，磷酸化或去除蛋白质的N端结构域均可实现激活。我们研究了Spo0A刺激spoIIG操纵子启动子转录的机制。该操纵子编码最早出现的区室特异性σ因子之一，其出现调控孢子形成的发育。当将激活的Spo0A与RNA聚合酶以及包含spoIIG启动子的DNA片段一起孵育时，相对于转录起始位点，-13至-3之间的碱基会发生变性。单独添加激活的Spo0A或RNA聚合酶不会诱导变性。在没有激活的Spo0A的情况下，两条链上在-3至-13位置之间均包含野生型启动子非模板序列的异源双链模板能够高效转录。这些数据表明DNA链分离是一个两步过程，并且Spo0A的激活产生了一种与聚合酶相互作用以诱导两步过程中第一步的形式。
