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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
由T7RNA聚合酶介导转录启动的结构转换
Srabani Mukherjee1, Luis G Brieba, Rui Sousa
1Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.
Cell
|August 2, 2002
概括
研究人员在转录启动过程中研究了T7RNA聚合酶. 他们发现了酶构成和DNA相互作用的特定变化,因为它从启动过渡到延长.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 遗传学 是一个遗传学.
背景情况:
- RNA聚合酶管理基因转录,这一过程涉及不同的启动和延长阶段.
- 从转录启动到延长的过渡涉及到RNA聚合酶构成及其与DNA相互作用的显著,但尚未被充分理解的变化.
- T7RNA聚合酶作为一个模型系统来研究这些基本的分子机制.
研究的目的:
- 描述T7RNA聚合酶在转录启动阶段的构造变化和DNA相互作用重排.
- 阐明聚合酶如何从促进体相互作用过渡到稳定延长的动态过程.
主要方法:
- 使用了T7RNA聚合酶,在11个不同的位置用Fe-BABE (N-(2-基乙烯) -1,2-乙二胺-N',N'',N''-三酸) 进行工程.
- 使用过氧化 (H2O2) 在修饰转录复合体的存在下诱导特定位点的DNA链裂变.
- 分析了DNA裂变的位置,以映射聚合酶-DNA接触和形状变化.
主要成果:
- 确定了核酸链分裂的特定地点,表明Fe-BABE合物与DNA的接近.
- 观察到的一系列分裂模式的变化,与转录启动的不同阶段相对应.
- 这些变化揭示了整个启动过程中蛋白质:DNA接触的动态重排.
结论:
- 该研究提供了在T7 RNA聚合酶转录启动过程中发生的过渡的详细分子地图.
- 证明了显著的形状灵活性和DNA接触的动态重新定位,因为聚合酶在启动过程中进展.
- 这些发现增强了我们对控制转录调节和酶-聚合物相互作用的基本机制的理解.
相关概念视频
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Bacterial Transcription
RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
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Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...

