環境因子調控大腸桿菌rne基因表現之研究

Abstract

mRNA 降解對於原核生物而言，是調節基因表現的重要機制。在大腸桿菌中，RNase E 對 mRNA 的降解以及 rRNA 熟成扮演了關鍵性的角色。RNase E 為細胞生長時必須的蛋白質，當其無活性時會造成大量 mRNA 半衰期延長與阻礙部分 mRNA 之降解，因此大腸桿菌利用回饋作用嚴格控制 RNase E 的生合成。此機制藉由 RNase E 作用於自身 mRNA 的 5’-untranslated region (UTR) 361個核苷酸來進行。 過去文獻指出，大腸桿菌 RNase E 的濃度及活性會隨生長環境不同而進行調整，為了得知環境因子如何在轉錄層面調控 rne 基因表現，我們利用插在染色體上的 rne-lacZ 活性以及北方墨點法來進行分析。至於RNase E的活性，則是藉由其受質 rpsO mRNA 的半衰期來測試。結果發現，rne mRNA 的表現量與 RNase E 的酵素活性，的確會受到不同碳源、生長速率與氧氣所影響。此外，亦發現 rne 基因表現受控於葡萄糖效應，當 LB 培養液外加 40mM 葡萄糖時， rne-lacZ 表現會下降。結果發現飢餓環境下，rne 基因表現降低會受 (p)ppGpp 調控。當大腸桿菌生長於有氧及無氧的環境下，轉錄因子 Integration host factor (IHF) 亦會對 rne 基因進行不同的調控方式。

mRNA degradation is an important mechanism of regulation gene expression in prokaryotes. In Escherichia coli, RNase E plays as a key role for mRNA degradation and rRNA maturation. This endonuclease is essential for cell growth. The inactivation of RNase E has been shown to prolong the lifetime of bulk mRNA and to impede the processing and decay of a variety of individual transcripts. E. coli cells tightly control RNase E synthesis through a feedback mechanism, which is mediated in cis by the 361-nucleotide rne mRNA 5’-untranslated region (UTR). Earlier reports have indicated that the intracellular RNase E concentration and its activity may change in response to modification of environmental conditions. To examine how the environmental factors regulated rne expression at transcription level, rne-lacZ operon fusions and Northern blotting were analyzed. In addation, rpsO mRNA, which is one of RNase E substrates was used to test RNase E activity. The results showed that the mount of rne mRNA and the activity of RNase E indeed changed in different carbon sources, growth rate and oxygen. Besides, rne expression was glucose repressed. After starvation, the expression of rne gene decressed because of (p)ppGpp effect. Finally, this study showed that integration host factor (IHF) regulated rne gene under aerobic and anaerobic growth conditions by different ways.