从结构到系统:RNA聚合酶II的高分辨率定量遗传分析
Hannes Braberg1, Huiyan Jin, Erica A Moehle
1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
Cell
|August 13, 2013
概括
研究人员绘制了RNA聚合酶II (RNAPII) 相互作用的地图,以了解基因表达. 他们发现转录速度影响拼接和开始部位选择,揭示了基因表达步骤的协调调节.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物化学 生物化学
背景情况:
- RNA聚合酶II (RNAPII) 是基因表达调节的核心.
- 了解RNAPII复杂的功能需要对其相互作用进行详细的分析.
研究的目的:
- 以功能地绘制RNAPII子域及其与其他蛋白质复合体的相互作用.
- 研究RNAPII聚合率,转录启动和拼接效率之间的关系.
主要方法:
- 使用53个RNAPII点突变生成了一个点突变表观小阵列概况 (pE-MAP).
- 在Saccharomyces cerevisiae中进行了定量遗传相互作用分析 (∼60,000个相互作用).
- 使用了拼接微阵列和体外延长率突变体.
主要成果:
- 启用了RNAPII子域的功能赋值,并确定了与蛋白质复合体的连接.
- 揭示了RNAPII速度和体内拼接效率之间的反向关系.
- 分类快速和缓慢的RNAPII突变影响起始地点选择 (上游与下游).
结论:
- 转录速率被微调,以调节多个基因表达步骤,包括启动和拼接.
- pE-MAP方法提供了一个强大的策略,用于在氨基酸分辨率下剖析多功能蛋白质机器.
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...
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...
Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
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...
Ribosome Profiling
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...

