相关实验视频
Updated: Jul 19, 2026

07:44
High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
概括
一个通用的转录因子BTF3对于准确的转录启动至关重要. 纯化的BTF3与RNA聚合酶B (II) 结合,形成一个活跃的转录复合体,而不会与DNA相互作用.
科学领域:
- 分子生物学分子生物学
- 基因转录 基因转录
- 蛋白质与蛋白质之间的相互作用
背景情况:
- 准确的转录启动对于基因表达至关重要.
- RNA聚合酶B (II) 是mRNA合成的中心酶.
- 一般的转录因子在转录中起着调节作用.
研究的目的:
- 为了净化和表征一般转录因子BTF3.
- 研究BTF3在转录启动中的作用.
- 确定BTF3与RNA聚合酶B (II) 和DNA的相互作用.
主要方法:
- 从HeLa细胞提取物中净化BTF3.
- 在体外转录试验中使用腺病毒-2主要晚期促进物 (Ad2MLP).
- 蛋白质-DNA和蛋白质-蛋白质相互作用的分析.
主要成果:
- BTF3被净化成一个27kDa的蛋白质.
- 对于Ad2MLP和其他RNA聚合酶B促进体的精确转录启动,BTF3是必要的.
- 纯化的BTF3与RNA聚合酶B (II) 结合,形成一个转录活性复合体.
- BTF3似乎不与DNA相互作用,也不对预启动复合体形成至关重要.
结论:
- BTF3是一种通用转录因子,直接与RNA聚合酶B (II) 相互作用.
- BTF3的功能包括通过聚合酶结合促进转录启动,而不是DNA相互作用.
- 这些发现阐明了调节真核细胞基因转录的关键机制.
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...
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...
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...
General Transcription Factors
Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
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...

