Shemiakin M F, Shumilov V Iu
Mol Biol (Mosk). 1981 Sep-Oct;15(5):1144-57.
The preincubation of a DNA with E. coli RNA polymerase provides its partial protection against the HindII, BspRI and AluI cleavage giving possibility to determine the location of RNA polymerase tight-binding sites. Using this approach about 16 RNA polymerase tight-binding sites were detected on replicative form of phiX174 phage DNA. The protection degree of each of these sites depended on the preincubation conditions. Some of the protected sites hit the known phiX174 promoters and rho-dependent terminators and the other were distributed along the whole phiX174 DNA molecule. Many of them could be considered as potential promoters because they contain all the necessary elements specifying the real promoter sequences. At least some of the intrinsic promoter elements could be observed next to the rest of protected sites. One of the protected sites (R6b/l) is located in phiX174 DNA region which is very similar to the cAMP-CRP-controlled promoter sequences. It was confirmed that phiX174 DNA has two B promoters positioned by Sanger on the phiX174 nucleotide map, according to our data obtained by RNA polymerase protection experiments along with RNA product analysis of the R8 DNA fragment transcription in vitro.
将DNA与大肠杆菌RNA聚合酶进行预孵育，可使其免受HindII、BspRI和AluI酶切的部分影响，从而有可能确定RNA聚合酶紧密结合位点的位置。采用这种方法，在phiX174噬菌体DNA的复制形式上检测到了约16个RNA聚合酶紧密结合位点。这些位点中每个位点的保护程度取决于预孵育条件。一些受保护的位点与已知的phiX174启动子和rho依赖性终止子重合，其他位点则沿整个phiX174 DNA分子分布。其中许多位点可被视为潜在的启动子，因为它们包含指定真实启动子序列的所有必要元件。在其余受保护位点旁边至少可以观察到一些内在启动子元件。其中一个受保护位点（R6b/l）位于phiX174 DNA区域，该区域与cAMP-CRP控制的启动子序列非常相似。根据我们通过RNA聚合酶保护实验以及体外R8 DNA片段转录的RNA产物分析获得的数据，证实了phiX174 DNA在Sanger绘制的phiX174核苷酸图谱上有两个B启动子。
