Lavialle C, Reuveni Y, Thoren M, Salzman N P
J Biol Chem. 1982 Feb 10;257(3):1549-57.
A detailed map of the initiation sites recognized by the Escherichia coli RNA polymerase (holoenzyme) on simian virus 40 (SV40) DNA has been constructed. An approach is described in order to compare the sites recognized on supercoiled as well as relaxed linear DNA. For localization of the initiation sites on supercoiled DNA, RNA polymerase-SV40 DNA Form I binary complexes are stabilized by incubation with three ribonucleoside triphosphates. The stable initiation complexes are cleaved with single cut restriction endonucleases in order to generate termination sites at precisely defined locations. Elongation of the RNA chains up to the cleavage site produces a defined set of discrete RNA species, whose size can be determined with accuracy by agarose gel electrophoresis following glyoxal denaturation. Each transcript is oriented by following two different experimental approaches. 1) The DNA of an SV40 deletion mutant is used as a template in comparison with that of wild type SV40; and 2) the RNAs synthesized after cleavage with different single cut restriction endonucleases are compared. Determination of the length and polarity of these RNAs allows us to accurately position the initiation sites on the physical map of the SV40 genome. A similar analysis was conducted for the mapping of the initiation sites recognized on precut linear SV40 DNA. Comparison of the two templates (supercoiled and linear) reveals that modification of the DNA conformation affects selection by the enzyme of certain promoters. Although most of the promoters are common to both templates, four are specifically recognized on superhelical DNA, while two others are used exclusively on linear SV40 DNA.
已构建出大肠杆菌RNA聚合酶（全酶）在猴病毒40（SV40）DNA上识别的起始位点的详细图谱。本文描述了一种方法，用于比较在超螺旋以及松弛线性DNA上识别的位点。为了定位超螺旋DNA上的起始位点，RNA聚合酶 - SV40 DNA I型二元复合物通过与三种核糖核苷三磷酸孵育来稳定。稳定的起始复合物用单切口限制性内切酶切割，以便在精确界定的位置产生终止位点。RNA链延伸至切割位点会产生一组确定的离散RNA种类，其大小可通过乙二醛变性后的琼脂糖凝胶电泳准确测定。每个转录本通过两种不同的实验方法来确定方向。1）将SV40缺失突变体的DNA与野生型SV40的DNA用作模板进行比较；2）比较用不同单切口限制性内切酶切割后合成的RNA。确定这些RNA的长度和极性使我们能够在SV40基因组的物理图谱上准确确定起始位点的位置。对预切割线性SV40 DNA上识别的起始位点进行了类似的图谱分析。比较这两种模板（超螺旋和线性）表明，DNA构象的改变会影响酶对某些启动子的选择。尽管大多数启动子在两种模板中都常见，但有四个在超螺旋DNA上被特异性识别，而另外两个仅在SV40线性DNA上被使用。
