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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
RNA聚合酶促进体开放复合物的结构组织
N Naryshkin1, A Revyakin, Y Kim
1Howard Hughes Medical Institute, Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
Cell
|July 13, 2000
概括
研究人员使用光交联绘制了细菌RNA聚合酶 (RNAP) 与促进者DNA的相互作用. 这张详细的地图揭示了RNAP促进器开放复合体 (RPo) 中的蛋白-DNA结合以及激活剂如何影响其结构.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 结构生物学 结构生物学
背景情况:
- 细菌RNA聚合酶 (RNAP) 对于转录启动至关重要.
- 在开放复合体 (RPo) 中理解RNAP促进器DNA相互作用是解读基因调节的关键.
- 之前的模型缺乏这些相互作用的详细原子级分辨率.
研究的目的:
- 为了精确地绘制RNAP促进器开放复合体 (RPo) 中的蛋白质-DNA相互作用.
- 基于实验交叉连接数据构建RPO的详细结构模型.
- 研究转录激活剂对RPo的结构影响.
主要方法:
- 采用系统的特定位置蛋白质-DNA光交联.
- 绘制了RNAP子单元和促进DNA酸盐之间的100多个不同的交叉链接.
- 利用交叉连接数据构建了一个高分辨率的RPo.Po的结构模型.
主要成果:
- 在RPo.com中提供了100多个蛋白质-DNA交叉链接的全面地图.
- 在特定RNAP段和DNA酸盐之间建立了详细的接触点.
- 能够构建一个精细的结构模型,用于具有催化能力的RPo.
结论:
- 这项研究为RNAP促进剂DNA相互作用提供了前所未有的详细视图.
- 生成的RPO结构模型作为理解转录启动的基础.
- 该方法允许分析转录激活剂如何调节RPO结构.
相关概念视频
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...
The Eukaryotic Promoter Region
The eukaryotic promoter region is a segment of DNA located upstream of a gene. It contains an RNA polymerase binding site, a transcription start site, and several cis-regulatory sequences. The proximal promoter region is located in the vicinity of the gene and has cis-regulatory sequences and the core promoter. The core promoter is the binding site for RNA polymerase and is usually located between -35 and +35 nucleotides from the transcription start site. The distal promoter regions are...
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:
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...
The Eukaryotic Promoter Region
The eukaryotic promoter region is a segment of DNA located upstream of a gene. It contains an RNA polymerase binding site, a transcription start site, and several cis-regulatory sequences. The proximal promoter region is located in the vicinity of the gene and has cis-regulatory sequences and the core promoter. The core promoter is the binding site for RNA polymerase and is usually located between -35 and +35 nucleotides from the transcription start site. The distal promoter regions are...

