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High-throughput Purification of Affinity-tagged Recombinant Proteins
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転写の構造的基礎:RNAポリメラーゼII活性センターの回転による核酸選択
Kenneth D Westover1, David A Bushnell, Roger D Kornberg
1Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Cell
|November 13, 2004
まとめ
RNAポリメラーゼIIは,活性部位の前にエントリー部位を含む,ニュクレオチド選択のための新しい2段階のメカニズムを使用します. この発見は,大規模で多サブユニットRNAポリメラーゼの理解を再定義する.
科学分野:
- 分子生物学は分子生物学である.
- 構造生物学 構造生物学とは
- バイオケミストリー バイオケミストリー
背景:
- RNAポリメラーゼIIは,真核生物の遺伝子転写に不可欠である.
- ニュクレオチド選択の正確なメカニズムを理解することは,遺伝子発現を調節する鍵です.
研究 の 目的:
- RNAポリメラーゼIIによるヌクレオチド選択のメカニズムを解明する.
- ヌクレオチド結合と結合の構造的基礎を特徴づける.
主な方法:
- X線結晶学を用いて,RNAポリメラーゼII転写複合体を視覚化しました.
- 酵素内のニュクレオチド結合部位と指向の分析.
主要な成果:
- エントリーサイト (E) と追加サイト (A) を含む2段階のヌクレオチド選択メカニズムが明らかにされました.
- 不一致した核酸は,潜在的な回転前にE部位で逆向きに結合する.
- 3番目の核酸結合部位は,潜在的にRNAの逆行を調節する;DNAの解き放たれは活性センターに先行する.
結論:
- この発見は,単一サブユニット酵素とは異なる,大規模なマルチサブユニットRNAポリメラーゼにおけるヌクレオチド選択のための新しいパラダイムを確立しています.
- この研究は,RNAポリメラーゼIIの機能と調節に関する原子レベルの洞察を提供します.
- 高解像度の結晶学は,酵素動力学とDNA相互作用の理解を深めた.
関連する概念動画
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...
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...

