Dombroski A J, Johnson B D, Lonetto M, Gross C A
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston 77030, USA.
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8858-62. doi: 10.1073/pnas.93.17.8858.
The promoters recognized by sigma 70, the primary sigma of Escherichia coli, consist of two highly conserved hexamers located at -10 and -35 bp from the start point of transcription, separated by a preferred spacing of 17 bp. sigma factors have two distinct DNA binding domains that recognize the two hexamer sequences. However, the component of RNA polymerase recognizing the length of the spacing between hexamers has not been determined. Using an equilibrium DNA binding competition assay, we demonstrate that a polypeptide of sigma 70 carrying both DNA binding domains is very sensitive to promoter spacing, whereas a sigma 70 polypeptide with only one DNA binding domain is not. Furthermore, a mutant sigma, selected for increasing transcription of the minimal lac promoter (18-bp spacer), has an altered response to promoter spacing in vivo and in vitro. Our data support the idea that sigma makes simultaneous, productive contacts at both the -10 and the -35 regions of the promoter and discerns the spacing between these conserved regions.
大肠杆菌的主要σ因子σ70所识别的启动子，由位于转录起始点上游-10和-35碱基对处的两个高度保守的六聚体组成，二者之间的最佳间距为17碱基对。σ因子有两个不同的DNA结合结构域，分别识别这两个六聚体序列。然而，RNA聚合酶中识别六聚体之间间距长度的组分尚未确定。通过平衡DNA结合竞争分析，我们证明携带两个DNA结合结构域的σ70多肽对启动子间距非常敏感，而仅具有一个DNA结合结构域的σ70多肽则不然。此外，一个为增加最小lac启动子（18碱基对间隔区）转录而筛选出的σ突变体，在体内和体外对启动子间距的反应都发生了改变。我们的数据支持这样一种观点，即σ因子在启动子的-10和-35区域同时进行有效的接触，并识别这些保守区域之间的间距。
