Ostrander E A, Benedetti P, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Science. 1990 Sep 14;249(4974):1261-5. doi: 10.1126/science.2399463.
Fusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components. The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site. Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription. Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences.
将酵母GAL4蛋白的DNA结合结构域与噬菌体T7 RNA聚合酶的氨基末端融合，可产生一种嵌合体，该嵌合体保留了其各组成部分的特性。GAL4肽的存在使嵌合酶能够将自身锚定在DNA模板上，当聚合酶位点处的RNA合成迫使DNA相对于该位点发生移位时，这种锚定反过来会驱动线性或环状模板中形成超螺旋DNA环。嵌合酶与DNA之间的非特异性相互作用似乎足以在转录过程中实现超螺旋化。嵌合聚合酶的转录严格依赖于T7启动子的存在；因此，它为在体外和体内特异性超螺旋化含有T7启动子序列的DNA片段提供了一种工具。
