Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins
Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins
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DOI：
10.1073/pnas.142002099
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发表时间：
2002-07-09
影响因子：
11.1
通讯作者：
McMacken, R
中科院分区：
文献类型：
--
作者：
Leng, FF;McMacken, R
在大肠杆菌中，RNA聚合酶转录可刺激局部DNA超卷曲。在体内，有广泛的实验支持“双结构域”模型，在该模型中，正DNA超级线圈在易位RNA聚合酶复合体前面产生，负超级线圈在其后面形成。负超级线圈在模板DNA中积累是因为正超级线圈被细胞拓扑异构酶优先去除。然而，在体外，对双结构域机制的明确和令人信服的支持一直缺乏。在这篇文章中，我们调和这种不一致，通过显示，在一个确定的体外系统与质粒DNA模板，各种序列特异性DNA结合蛋白，如噬菌体λ O复制启动物或大肠杆菌乳糖或半乳糖抑制物，显著刺激转录偶联DNA超螺旋。我们进一步证明，这种刺激需要在DNA模板中存在相关DNA结合蛋白的识别序列，并依赖于RNA聚合酶产生的长RNA链。我们的数据与特定DNA结合蛋白促进双结构域机制在转录过程中增强DNA超卷曲的模型最为一致。更准确地说，我们认为一些核蛋白复合物，可能是那些含有急剧弯曲DNA的核蛋白复合物，可以形成屏障，阻碍独立染色体超级螺旋结构域的扩散和合并。核蛋白复合物对DNA超螺旋的定位可能是调节对DNA超螺旋敏感的DNA交易的一般机制。
Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a "twin-domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twin-domain mechanism has been lacking. in this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNA-binding proteins, such as the bacteriophage lambda O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.