Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase.
Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase.
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DOI：
10.1126/science.2399463
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发表时间：
1990-09
期刊：
影响因子：
56.9
通讯作者：
Elaine A. Ostrander;P. Benedetti;JC Wang
中科院分区：
文献类型：
--
作者：
Elaine A. Ostrander;P. Benedetti;JC Wang
酵母GAL 4蛋白的DNA结合域与噬菌体T7 RNA聚合酶的氨基末端的融合产生保留其组分特征的嵌合体。GAL 4肽的存在允许嵌合酶将其自身锚在DNA模板上，并且当聚合酶位点处的RNA合成迫使DNA相对于该位点移位时，这种锚定又驱动线性或环状模板中超螺旋DNA环的形成。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影响超螺旋。通过嵌合聚合酶的转录严格依赖于T7启动子的存在;因此，它提供了用于特异性超螺旋化含有T7启动子序列的DNA片段的体外和体内工具。
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.