研究 RprA sRNA 在大腸桿菌內之調控角色

Abstract

在大腸桿菌中，RprA 為全長 105 個核苷酸之非轉譯小型核醣核酸。稍早的 研究發現，RprA 會在細胞生長平原期 (stationary phase) 或是高透壓環境中時大 量表現，並藉由與目標 mRNA 之 5’-UTR 鹼基配對 (base-pairing) ，影響 RNA 的 穩定度或轉譯效率。目前已知受 RprA 調控的基因有 rpoS 、 csgD 以及 ydaM。 為了探討 RprA 在高滲透壓所扮演的調控角色，我們利用生物晶片研究 RprA 大 量表現 (overexpression) 對大腸桿菌全轉錄體之影響，並利用即時聚合酶連鎖反 應 (real-time PCR) 進一步驗證。透過上述實驗，我們發現 10 個基因的 RNA 表現 量隨著 RprA 表現而改變，表示 RprA 可能直接調控此群基因表現。這些基因與 碳源代謝以及細胞膜通道有關，由此推論 RprA 反應胞外刺激，透過調節膜蛋白 及代謝作用以應對高滲透壓環境。我們利用 RNAhybrid 預測 RprA 與 lamB mRNA 5’端之鹼基配對位置 (base-pairing site)，並利用綠螢光轉譯報導系統 (GFP translational fusion) 與突變實驗，證實 RprA 負向調控 lamB 轉譯效率。在上述基 因中，lamB 產物為 maltoporin，除了參與 maltose 代謝外，也是 lambda 噬菌體 之接受器。並進一步造成 lambda 噬菌體感染力下降。最後，彙整上述受 RprA 調控的基因、相關轉錄因子與其生理意義，重建在高滲透壓環境下，RprA sRNA 所 參與的基因調控網絡，並釐清 RprA 所扮演的調控角色。

Recent studies revealed that bacteria encode a tremendous number of small non-coding RNA (sRNA). In Escherichia coli, RprA is a 105 nt long sRNA. Previous studies have shown that RprA sRNA accumulates in the stationary phase in response to high osmolality. By base-pairing with the 5’-untranslated region of the target mRNA, it regulates the mRNA of rpoS, csgD, and ydaM in RNA stability or during protein translation. To investigate the regulatory role of RprA under high osmotic stress, a genome-wide transcriptome that overexpresses the rprA gene was compared with an rprA deletion strain using a microarray. The results were then confirmed via real-time PCR, and 10 genes were regulated using RprA sRNA in a time course, which suggests that they were direct targets of RprA sRNA. These genes are involved in carbon metabolism and membrane stress. The results indicate that RprA links the outer stimuli and central metabolism together to adjust cellular physiology in response to osmotic stress. The base-pairing site of RprA/target was predicted using RNAhybrid. The base-pairing site was tested with GFP translational fusion and was validated using rprA sequence deletion. We demonstrated that the lamB gene that encodes the phage receptor maltoporin is a novel and negatively controlled RprA target. By adapting these direct targets and their physiologic roles, we reconstructed the network governed by RprA sRNA to elucidate the mechanism of gene regulation in response to high osmolality.