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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
转录的结构基础:以3.4安格斯特罗姆分辨率的逆向RNA聚合酶II
Dong Wang1, David A Bushnell, Xuhui Huang
1Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
概括
研究人员确定了RNA聚合酶II在逆向状态下的晶体结构,揭示了对校对和RNA裂变至关重要的特定结合部位. 这一发现提升了我们对转录忠实性的理解.
科学领域:
- 分子生物学分子生物学
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- RNA聚合酶 (RNAPs) 是必要的酶,可以将DNA转录为RNA.
- 在转录过程中,RNAP通过多个构造状态进行动态循环.
- 之前的结构研究阐明了RNAPs的转移前和转移后状态.
研究的目的:
- 为了确定RNA聚合酶II在逆转位 (回溯) 状态中的晶体结构.
- 描述逆行状态的结构特征,包括核酸结合点.
- 调查延长因子SII (TFIIS) 在促进RNA分裂的作用.
主要方法:
- 使用X射线晶体学来确定RNA聚合酶II的结构.
- 在回溯状态下实现了RNA聚合酶II的结晶.
- 与延长因子TFIIS进行了联合结晶,以捕获准备分裂的复合物.
主要成果:
- 在逆行状态下成功确定了RNA聚合酶II的晶体结构.
- 一个特定的核酸结合位点,称为"P"位点,在回溯结构中被确定.
- 结构显示TFIIS结合时发生了重新排列,将RNA定位为分裂.
结论:
- RNA聚合酶II的逆向状态具有独特的P位点,用于结合错误的核酸或受损的DNA.
- 这个P位点是转录的校对机制的组成部分.
- 结合TFIIS的结构为RNA裂变的机制提供了洞察力,提高了转录保真性.
相关概念视频
Transcription Initiation
Initiation is the first step of transcription in eukaryotes. Prokaryotic RNA Polymerase (RNAP) can bind to the template DNA and start transcribing. On the other hand, transcription in eukaryotes requires additional proteins, called transcription factors, to first bind to the promoter region in the DNA template. This binding helps recruit the specific RNAP that can assemble on the DNA and start transcription.
The promoters and enhancers and their accessory proteins allow tight regulation of...
The promoters and enhancers and their accessory proteins allow tight regulation of...
Eukaryotic RNA Polymerases
RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the...
All three eukaryotic RNAPs require specific transcription factors, of which the...
Eukaryotic RNA Polymerases
RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the...
All three eukaryotic RNAPs require specific transcription factors, of which the...
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.
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
RNA Polymerase II Accessory Proteins
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...
RNA Polymerase II Accessory Proteins
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...

