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首页> 外文期刊>Molecular and Cellular Biology >The ZEBRA activation domain: modular organization and mechanism of action.
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The ZEBRA activation domain: modular organization and mechanism of action.

机译：ZEBRA激活域：模块化组织和作用机制。

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An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers. The regulatory significance of this parallel organization has not been systematically explored. We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA. We performed our experiments in vitro so that the activator concentrations, stabilities, and affinities for DNA could be monitored. ZEBRA and various amino-terminal deletion derivatives, expressed in and purified from Escherichia coli, were assayed in a HeLa cell nuclear extract for the ability to activate model reporter templates bearing one, three, five, and seven upstream ZEBRA binding sites. Our data show that ZEBRA contains four modules that contribute to its potency in vitro. The modules operate interchangeably with promoter sites to determine the transcriptional response such that the loss of modules can be compensated for by increasing promoter sites. Potassium permanganate footprinting was used to show that transcriptional stimulation is a consequence of the activator's ability to promote preinitiation complex assembly. Kinetic measurements of transcription complex assembly in a reconstituted system indicate that ZEBRA promotes formation of a subcomplex requiring the TFIIA and TFIID fractions, where TFIIA acts as an antirepressor. We propose a model in which the concentration of DNA-bound activation modules in the vicinity of the gene initiates synergistic transcription complex assembly.
机译：RNA聚合酶II激活剂通常包含几个有助于其效力的区域，表面上类似于启动子和增强子的模块化结构。该平行组织的监管意义尚未得到系统地探讨。我们通过检查爱泼斯坦-巴尔病毒反式激活因子ZEBRA的激活域来考虑这个问题。我们在体外进行了实验，以便可以监测活化剂的浓度，稳定性和对DNA的亲和力。在HeLa细胞核提取物中检测并表达了从大肠杆菌中表达并纯化的ZEBRA和各种氨基末端缺失衍生物，以激活带有一个，三个，五个和七个上游ZEBRA结合位点的模型报告模板的能力。我们的数据表明，ZEBRA包含四个有助于其体外效力的模块。所述模块与启动子位点可互换地操作以确定转录应答，从而可以通过增加启动子位点来补偿模块的损失。高锰酸钾足迹被用来表明转录刺激是活化剂促进预起始复合物组装的能力的结果。重构系统中转录复合物装配体的动力学测量表明，ZEBRA促进了需要TFIIA和TFIID组分的亚复合物的形成，其中TFIIA充当抗阻遏物。我们提出了一个模型，其中该基因附近的DNA结合激活模块的浓度启动了协同转录复合体装配。

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