Regulation of Gene Transcription

我们以大肠杆菌中的半乳糖酶操纵子为模型系统，研究了转录起始受激活子和阻遏子调控的机制。gal操纵子由两个串联启动子P1和P2转录，这两个串联启动子由许多调节蛋白以多种方式调节，这些调节蛋白通过结合到gal DNA上的相应位点而起作用。我们报告了去年的几个重大发现。 gal转录的主要调控方式是通过形成一种称为阻遏体的DNA-多蛋白复合物来实现的，该复合物导致两种启动子的阻遏。在大肠杆菌中，当两个二聚体GalR蛋白和组蛋白样HU蛋白结合到引起DNA成环的同源位点时，发生Gal阻遏体组装和Gal操纵子的阻遏。以前的实验表明，DNA环包含113 bp的启动子区域。如果DNA上的结合位点正确对齐，则允许两个DNA结合蛋白质之间的相互作用。要测试的想法，即观察到的抑制在体外半乳糖转录介导的DNA环，我们研究了改变的两个GalR结合位点，OE和OI的DNA螺旋的相对角方向的影响。我们发现，抑制是一个周期性的函数的两个操作网站之间的距离。由于阻遏复发相称的DNA螺旋重复序列，我们得出结论，在体外观察到的阻遏是介导的DNA循环和在体外条件下反映在体内的情况。 基于结构的遗传分析定义了GalR表面相互作用形成堆叠的V形四聚体结构。立体化学模型的四个可能的DNA环兼容的GalR四聚体的配置构建使用序列依赖的结构参数的互操作DNA和构象变化所造成的GalR和不对称HU结合。他们的DNA弹性能的评估明确的优先权的循环结构中，两个半乳糖经营者采取反平行的方向，导致undertwiring的DNA。 我们还研究了HU结合DNA对GalR结合两个运营商（协同性）的依赖性。我们发现GalR通过特定的GalR-HU相互作用将HU背到DNA上的关键位置。这是HU与另一种蛋白质特异性接触的第一个例子。GalR-HU接触导致两种蛋白质与DNA的合作结合可能是短暂的，并且在最终的阻遏体结构中不存在。通过与调节蛋白的特异性缔合而被募集到DNA上的结构位点的序列非依赖性DNA结合蛋白可能是组装复杂核蛋白结构的常见模式。

We have been studying the mechanism by which initiation of transcription is regulated by activators and repressors, by using the galactase operon in Escherichia coli as a model system. The gal operon is transcribed by two tanden promoters, P1 and P2, which are regulated in a variety of ways by a number of regulatory proteins, which act by binding to their corresponding sites on the gal DNA. We report several critical discoveries made last year. The major mode of regulation of gal transcription is achieved by formation of a DNA-multiprotein complex, called repressosome, which causes repression of both promoters. Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping. Previous experiments suggested that the DNA loop contains 113 bp encompassing the promoter region. Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned. To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of the two GalR binding sites, OE and OI in the DNA helix. We found that repression is a periodic function of the distance between the two operator sites. Since repression recurred commensurate with DNA helical repeat, we concluded that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation. Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure. Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding. Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA. We also investigated the dependency of HU binding to DNA on GalR binding to the two operators (cooperativity). We showed that GalR piggybacks HU to the critical position on the DNA through a specific GalR-HU interaction. This is the first example of HU making a specific contact with another protein. The GalR-HU contact resulting in cooperative binding of the two proteins to DNA may be transient and absent in the final repressosome structure. A sequence- independent DNA-binding protein being recruited to an architectural site on DNA through a specific association with a regulatory protein may be a common mode for assembly of complex nucleoprotein structures.