Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping
Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping
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DOI：
10.1073/pnas.2034851100
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发表时间：
2003-09-30
影响因子：
11.1
通讯作者：
Finzi, L
中科院分区：
文献类型：
--
作者：
Lia, G;Bensimon, D;Finzi, L
DNA 的整体拓扑深刻地影响转录的调节，并由 DNA 灵活性以及诱导 DNA 扭转、扭曲和/或成环的蛋白质结合决定。 Gal阻遏物(GaIR)被认为通过形成包围启动子的约40nm DNA的DNA环来抑制大肠杆菌gal操纵子的两个启动子的转录。转录抑制的拓扑调控机制的相关证据是需要超螺旋 DNA 模板和类组蛋白热不稳定核蛋白 (HU)。通过使用单分子操作来产生和微调 DNA 分子中的张力，我们直接检测到 GaIR/HU 介导的 DNA 环，并表征了其动力学、热力学和超螺旋依赖性。单分子实验中 gal DNA 成环所需的因素（HU、GalR 和 DNA 超螺旋）与体外和体内观察到的 gal 抑制所需的因素完全一致。我们的单电子分子实验表明，负超螺旋 DNA 在轻微张力下会变性，促进 GaIR/HU 介导的 DNA 环形成。这种拓扑中间体在其他转录、复制和重组的多蛋白复合物中可能具有类似的作用。
The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GaIR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of approximate to40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GaIR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GaIR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our sing e-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GaIR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.