Chen Y F, Helmann J D
Section of Microbiology, Cornell University, Ithaca, New York 14853, USA.
J Mol Biol. 1995 Jun 16;249(4):743-53. doi: 10.1006/jmbi.1995.0333.
Flagellar biosynthesis requires an alternative sigma (sigma) subunit of RNA polymerase to allow recognition of the promoters for flagellin and other late genes of the flagellar regulon. We have now overproduced and characterized Bacillus subtilis sigma D: the prototype of the sigma 28 family of flagellar sigma factors. Limited protease digestion studies indicate that sigma D contains an amino-terminal domain, comprising conserved regions 1.2 and 2, and a carboxyl-terminal domain containing conserved regions 3.2 and 4. The protease-sensitive region between these two domains correlates with a region of very low sequence conservation among bacterial sigma factors. Unlike the primary sigma factor, sigma D binds to DNA. In non-denaturing polyacrylamide gel electrophoresis the sigma D-DNA complex has an apparent equilibrium dissociation constant of 1 microM. Binding of sigma D to the promoter for flagellin, PD-6, appears to lead to an altered DNA structure near the -35 and -10 recognition elements as detected by DNase I footprinting and by the enhanced reactivity of several bases to dimethylsulfate.
鞭毛生物合成需要RNA聚合酶的一种替代σ（sigma）亚基，以便识别鞭毛蛋白启动子和鞭毛调节子的其他晚期基因。我们现已过量表达并鉴定了枯草芽孢杆菌σD：鞭毛σ因子σ28家族的原型。有限蛋白酶消化研究表明，σD包含一个氨基末端结构域，由保守区域1.2和2组成，以及一个羧基末端结构域，包含保守区域3.2和4。这两个结构域之间对蛋白酶敏感的区域与细菌σ因子中序列保守性非常低的一个区域相关。与主要的σ因子不同，σD能与DNA结合。在非变性聚丙烯酰胺凝胶电泳中，σD-DNA复合物的表观平衡解离常数为1微摩尔。通过DNase I足迹法以及几个碱基对硫酸二甲酯反应性增强检测发现，σD与鞭毛蛋白启动子PD-6的结合似乎导致了-35和-10识别元件附近DNA结构的改变。
