トランスドミナントRNAポリメラーゼ変異によって,イニシアーションから延長への移行をブロックする
M Kashlev1, J Lee, K Zalenskaya
1Institute of Molecular Genetics, U.S.S.R. Academy of Sciences, Moscow.
まとめ
Escherichia coliのRNAポリメラーゼの突然変異は,DNAの延長を防ぐことによって遺伝子発現を妨害する. この変異酵素は細胞の成長を阻害し,プロモーターへのワイルド型ポリメラーゼのアクセスを阻害します.
科学分野:
- 分子生物学は分子生物学である.
- 酵素学 酵素学とは
- 遺伝学 遺伝学とは
背景:
- RNAポリメラーゼは遺伝子発現に不可欠であり,構造的特徴が保存されています.
- RNAポリメラーゼのβサブユニットは,その触媒機能において重要な役割を果たします.
研究 の 目的:
- Escherichia coli RNAポリメラーゼのβサブユニットにおける進化的に不変のアミノ酸置換 (Lys1065Arg) の機能的影響を調査する.
- この変異がRNAポリメラーゼの活性,細胞の成長,および転写にどのように影響するかを決定する.
主な方法:
- サイト指向型ミュータゲネシスにより,E. coli RNAポリメラーゼのβサブユニットにLys1065Arg置換を導入する.
- 誘導可能なプロモーターからの変異RNAポリメラーゼの発現.
- ホロ酵素の組み立て,プロモーター複合体の形成,ディヌクレオチド合成,および転写延長の分析.
- 細胞成長と野生型酵素活性に対する変異性ポリメラーゼの効果の評価.
主要な成果:
- ベータサブユニットのLys1065Arg置換は,RNAポリメラーゼの触媒センターを破壊した.
- 変異タンパク質の発現は細胞の成長を阻害する.
- 変異したホロ酵素は,安定したプロモーター複合体を形成し,ディヌクレオチドを合成したが,延長を開始することはできなかった.
- 変異したポリメラーゼは,野生型のRNAポリメラーゼがプロモーターにアクセスするのを防ぐことによって転写を阻害しました.
結論:
- ベータサブユニットの進化的に不変のLys1065残基は,RNAポリメラーゼの触媒活性と転写延長に決定的な役割を果たしています.
- この残留物の破壊は,変異型および野生型ポリメラーゼの機能を抑制し,細胞の生命力に影響を及ぼし,支配的な負の効果をもたらします.
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