碳源與核酸內切酶對大腸桿菌延胡索酸酶 fumA、fumC 基因表現之研究

Abstract

本實驗室先前研究結果發現，fumA 與 fumC 的 mRNA 穩定性會隨著細菌生長速率上升而增加。本研究中，我們將大腸桿菌培養在不同碳源下，分別觀察在轉錄、轉錄後與轉譯三個層次 fumA 基因表現情形。為探究影響 mRNA 穩定性的因子為何，我們使用連續式培養來觀察核糖核酸內切酶 RNase E 與 poly(A) 聚合酶在不同生長速率下，對 fumA 與 fumC mRNA 穩定性的影響。 結果顯示在轉錄層次，以醋酸為碳源時，fumA mRNA 表現量比以葡萄糖為碳源時較多。在轉錄後層次，以葡萄糖為碳源時，fumA mRNA 較以醋酸為碳源時穩定。在轉譯層次，以葡萄糖為碳源時，FumA 蛋白質比以醋酸為碳源時表現量較多。在連續式培養下，fumA 與 fumC mRNA 穩定性會隨著細菌生長速率上升而增加。當細胞內 RNase E 突變後，fumA 與 fumC mRNA 皆比野生株明顯穩定，但仍受生長速率的調控。當 poly(A) 聚合酶缺失後，fumA 與 fumC mRNA 的穩定性與野生株比較後並沒有顯著改變。當 RNase E 與 poly(A) 聚合酶同時缺失，fumA 與 fumC mRNA 的穩定性則大幅提昇，比 RNase E 單獨缺失時更穩定。此研究結果顯示 fumA 與 fumC 基因在轉錄、轉錄後與轉譯均受碳源及生長速率所調控。

The previous studies showed that fumA and fumC mRNA stability increased with cell growth rate. In this study, we examined the effect of different carbon sources on fumA gene expression in transcription, post-transcription, and translation level, and used continuous culture to examine the effect of RNase E (rne) and poly (A) polymerase I (pcnB) on fumA and fumC mRNA degradation under different growth rates in E. coli. The results of this study demonstrated that fumA mRNA amount was higher in acetate medium than in glucose medium at transcription level. In post-transcription level, fumA mRNA was more stable in glucose medium than in acetate medium. In translation level, FumA protein was higher in glucose medium than in acetate medium. In continuous culture, both fumA and fumC mRNA stability increased with cell growth rate. The stability of fumA and fumC mRNA in rne－ strain were higher than in wild type strain. There were no significant change of fumA and fumC mRNA stability in pcnB－ strain. In rne－pcnB－ double mutant, fumA and fumC mRNA stability were higher than in rne－ strain. These results indicated that fumA and fumC gene expression were regulated by carbon sources and growth rates at transcription, post-transcription, and translation levels.