相关实验视频
Updated: Jul 15, 2026

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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
一个编码RAP74的cDNA,它是RNA聚合酶II转录的一般启动因子
A Finkelstein1, C F Kostrub, J Li
1Department of Biochemistry, Michigan State University, E. Lansing 48824.
Nature
|January 30, 1992
概括
研究人员分离了人类RAP74的cDNA,这是转录因子RAP30/74 (TFIIF) 的关键组成部分. 这一因素对于RNA聚合酶II预启动复合体的形成和精确的转录启动至关重要.
科学领域:
- 分子生物学分子生物学
- 基因规则 基因规则
- 生物化学 生物化学
背景情况:
- RNA聚合酶II需要一般的转录因子来启动.
- RAP30/74 (TFIIF) 是一个通用转录因子,对于预启动复杂组合至关重要.
- RAP30子单元与细菌的西格玛因子具有同质性.
研究的目的:
- 为了分离编码人类RAP74亚单元的互补DNA (cDNA).
- 描述RAP30和RAP74在转录启动中的作用.
- 评估重组RAP30和RAP74在转录试验中的有用性.
主要方法:
- 补充DNA (cDNA) 的隔离和测序.
- 在大肠杆菌中的重组蛋白表达.
- 使用RNA聚合酶II的体外转录试验.
主要成果:
- 人类RAP74cDNA的分离.
- 证明RAP30和RAP74都是预启动复合体的组成部分.
- 再组合的人类RAP30和RAP74可以在转录启动过程中功能性地替代自然复合物.
结论:
- 人类RAP74cDNA的分离为进一步研究TFIIF提供了一个工具.
- 重组TFIIF在体外具有功能,使得详细的机制研究成为可能.
- 了解TFIIF的功能对于破译RNA聚合酶II对基因转录的调节至关重要.
相关概念视频
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...
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...
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...
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...

