構造からシステムへ:RNAポリメラーゼIIの高解像度の定量遺伝分析
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
まとめ
研究者は遺伝子発現を理解するためにRNAポリメラーゼII (RNAPII) の相互作用をマッピングしました. 彼らは,転写速度がスプライシングと開始部位の選択に影響することを発見し,遺伝子発現のステップの調整された規制を明らかにしました.
科学分野:
- 分子生物学は分子生物学である.
- 遺伝学 遺伝学とは
- 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 (RNAPII) は,遺伝子発現の調節に中心的な役割を果たしています.
- RNAPIIの複雑な機能を理解するには,その相互作用の詳細な分析が必要です.
研究 の 目的:
- RNAPIIサブドメインとその他のタンパク質複合体との相互作用を機能的にマッピングする.
- RNAPIIポリメリゼーション率,転写開始,およびスプライシング効率の間の関係を調査する.
主な方法:
- 53のRNAPII点変異体を使用して,点変異性エピスタティックミニアレイプロファイル (pE-MAP) を生成しました.
- Saccharomyces cerevisiae.で定量的な遺伝子相互作用分析 (∼60,000の相互作用) を実施しました.
- スプライシングマイクロアレイとインビトロ伸縮率変異体を使用した.
主要な成果:
- RNAPIIサブドメインの機能的割り当てを有効にし,タンパク質複合体との接続を特定しました.
- RNAPIIの速度とin vivoのスプライシング効率の間の逆関係が明らかになった.
- 開始部位選択に影響を与える分類された高速および遅いRNAPII変異体 (アップストリーム対ダウンストリーム).
結論:
- 転写速度は,イニシアーションとスプライシングを含む複数の遺伝子発現のステップを調節するために微調整されています.
- pE-MAPアプローチは,アミノ酸解像度で多機能タンパク質マシンを解剖するための強力な戦略を提供します.
関連する概念動画
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...

