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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
在RNA聚合酶II的非酸化形式和TATA结合蛋白之间的特定相互作用
A Usheva1, E Maldonado, A Goldring
1Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Cell
|May 29, 1992
概括
RNA聚合酶II的碳氧终端域 (CTD) 对于转录至关重要. 它与TATA结合蛋白 (TBP) 的相互作用对于启动基因转录至关重要,酸化影响了这种结合.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 基因规则 基因规则
背景情况:
- RNA聚合酶II是转录蛋白质编码基因的中心酶.
- RNA聚合酶II的碳氧终端域 (CTD) 在转录中起着调节作用.
- 塔塔结合蛋白 (TBP) 是一种关键的转录因子,在II类促进体中需要启动转录.
研究的目的:
- 为了研究RNA聚合酶II CTD 复合体重复在转录活性中的作用.
- 为了确定RNA聚合酶II和TATA结合蛋白 (TBP) 之间的相互作用.
- 阐明CTD调解与TBP相互作用的机制.
主要方法:
- 亲和染色法使用列与RNA聚合酶II CTD.不动化的heptamer重复.
- 细分HeLa细胞提取物以分离转录活性成分.
- 测试用于测量转录活性和蛋白质与蛋白质相互作用.
主要成果:
- 当HeLa细胞提取物在含有野生类型CTD七度重复体的列上分成时,失去了转录活性.
- 这种活动丧失是野生类型重复的特征,因为突变版本没有影响.
- 通过添加TFIID,TBP或从列中溶解的蛋白质来恢复转录活性,这表明TBP的参与.
- RNA聚合酶II在非酸化形式特别结合TBP,而酸化形式没有相互作用.
- 确定了RNA聚合酶II的CTD作为与TBP的特定相互作用的调解因素.
结论:
- CTD 七度重复对于保持RNA聚合酶II的转录能力至关重要.
- RNA聚合酶II和TBP之间的相互作用对转录启动至关重要,并由CTD调解.
- RNA聚合酶II的酸化状态影响其与TBP相互作用的能力,这表明它在转录中起着调节作用.
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