大腸桿菌grxD基因表現之調控

Abstract

本研究探討大腸桿菌之 grxD 基因在菌體內所受到的調控。藉由不同長度的 grxD 基因上游未轉譯區，與 lacZ 基因進行 operon 及 protein fusion。首先，發現 grxD-lacZ 融合基因於對數期的表現量逐漸增加，並於靜止期前達到最高量，繼而稍降維持平穩表現。將預測之啟動子 P1grxD 的 -35 位置及 P2grxD 的 -10 處突變後，可發現此融合基因的表現量均會降低，故推測 grxD 基因表現受到一個以上的啟動子調控。而上游啟動子 P1grxD 主導對數期表現量的增加，下游啟動子 P2grxD 則維持基因的基本表現。 厭氧狀態時，此融合基因表現量下降，且兩個啟動子均受到抑制。但將保守區域 UCR 刪除後，其於厭氧狀態時的表現量提升；又在厭氧狀態下， fnr- 缺失突變株的表現量不會受到抑制。故推測 grxD 的基因表現於厭氧狀態下，受到調控因子 FNR 直接的抑制，且上游序列包含了一個以上的 FNR cis-binding site 。 本研究於 fur- 、 ryhB- 及此二基因的雙重缺失的情況下，發現 grxD-lacZ 融合基因的表現固然受到 small RNA RyhB 的抑制，但轉錄因子 Fur 仍具有正向調控的作用，甚至是在鐵缺失的情況下。則當刪除 UCR 區域後，不論是於LB 培養基或鐵缺失狀態下，RyhB 均不會影響融合基因的表現量，所以此段 UCR 區域有可能為 RyhB 的 binding site。 除此之外，為了瞭解 Grx4 扮演的角色，我們使用酵母菌雙雜交系統測試了可能有交互作用產生的對象。於細胞體內 (in vivo) 我們證實了 Grx4 與 Grx1 及 TrxB 可能存在的交互作用。此外還發現 Grx4 自己本身，以及與 BolA 之間有很強的交互作用現象。

This study was investigated the regulation of grxD gene expression in Escherichia coli. Based on different lengths of promoter fragment in grxD-lacZ operon and protein fusion, we have found that the gene expression was increased upon entry into stationary phase, and the expression of grxD was enhanced by two promoters P1grxD and P2grxD. While either promoter mutated at the location of -35 (P1grxD) and -10 (P2grxD) respectively, the promoter activity was decreased. In log phase, the grxD was more activated with P1grxD promoter than that with P2grxD. Under anaerobic condition, the grxD-lacZ gene expression is reduced with both promoters P1grxD and P2grxD. However, it is up-regulated with the one carrying a deletion of unknown conserved region (UCR). In fnr- strain, the expression of grxD-lacZ gene is increased and we suggest that the grxD gene expression is directly repressed by FNR. These findings implied that more than one FNR cis-acting sites are present in the promoter region of grxD. In fur-, ryhB- or double mutant, we found that the grxD-lacZ gene expression is reduced by RyhB, but Fur still up-regulates the expression even upon iron depletion. When deleted the UCR region, RyhB do not affect the expression level even under iron depletion or LB condtion. We suggest that the UCR region is a RyhB binding site. Furthermore, we use yeast two-hybrid method to reveal the Grx4 function in vivo. We confirmed that Grx4 interact with Grx1 and TrxB in vivo, and also found Grx4 interact strongly with itself and BolA, and slightly interact with Grx2 and TrxA.