CRP蛋白調控大腸桿菌atpIBEFHAGDC操縱子之研究

Abstract

腺苷三磷酸（ATP）是一種重要的能量轉移分子，大腸桿菌內atpIBEFHAGDC操縱子會轉譯成FoF1 ATP酶複合物，它可在氧化磷酸化代謝途徑中催化細胞內ATP合成或分離。 環磷酸腺苷AMP受體蛋白（CRP）是一個廣泛的轉錄因子，在大腸桿菌中已知調控超過423個基因。先前的研究證實，大腸桿菌細胞能量狀態與CRP調控磷酸甘油酸激酶（pgk）、琥珀酰硫激酶（sucCD）、細胞色素C氧化酶（cyoABCDE）和琥珀酸脫氫酶（sdhCDAB）的代謝途徑相關。 本研究主要目的是證實CRP蛋白在大腸桿菌內會對atpIBEFHAGDC操縱子在轉錄層次進行調控作用。在研究中我們利用電泳遷移率變動分析(electrophoretic mobility shift assay, EMSA)及足跡法(footprinting)來驗證atp操縱子啟動子區的CRP結合序列。進一步利用即時定量real-time PCR和 lacZ reporter fusion的技術，來驗證CRP對atp操縱子的調控關係。實驗結果證實CRP蛋白在轉錄層次中會活化所有atp操縱子的基因表現。微陣列分析結果也證實atp 操縱子主要是受到atpIp啟動子所調控。 此外，為了證實CRP對atp操縱子在生理層次上也有調控作用，藉由構築atpI啟動子區CRP的結合序列的突變株。比較了大腸桿菌野生型和突變株中ATP、ADP的產量及ATP/ ADP比值，結果證實CRP扮演一個協調者可調控細胞的能量狀態。CRP蛋白藉由調控atp操縱子的ATP生成量來調控能量狀態，因而間接影響轉移RNA來調控蛋白質表現量。

Adenosine-triphosphate (ATP) is an important energy transfer molecule. The atpIBEFHAGDC operon encodes the FoF1 ATPase complex, which catalyzes the metabolism of cellular ATP involved in oxidative phosphorylation in Escherichia coli. Cyclic–AMP receptor protein (CRP) is a global transcription factor that regulates more than 423 genes in E. coli. Previous studies indicated that CRP regulates phosphoglycerate kinase (pgk), succinyl thiokinase (sucCD), cytochrome C oxidase (cyoABCDE), and succinate dehydrogenase (sdhCDAB) in the metabolic pathway correlated with the cellular energy state in E. coli. cAMP levels are low in E. coli cultured in glucose-supplemented media. Meanwhile, the expression of atpI is lower in glucose-supplemented Luria–Bertani (LB) medium than in pure LB. This study aims to verify the regulatory function of CRP in the transcriptional regulation of the atpIBEFHAGDC operon in E. coli under various conditions. The first gene of the atp operon is atpI. The CRP binding site of the atpI promoter region was confirmed through electrophoretic mobility shift assay and DNase I foot-printing assay. The regulatory roles of CRP in the atp operon were further examined by real-time PCR and lacZ reporter fusion. Our results indicated that CRP is an activator of all genes of the atp operon. Consistent results were obtained from the cAMP dose-response assay. The microarray analysis results also suggested that the atp operon is generally regulated by the atpIp promoter. Furthermore, the CRP binding site on the atpI promoter region was mutated through counter-selection to verify the physiological significance of CRP-regulated atpI operon. The evidence of growth curve, ATP, ADP yields, and ATP/ADP ratio in E. coli wild-type and mutant strains indicated that CRP serves as a coordinator in increasing the cellular energy state. The CRP of the atp operon regulates the ATP level in energy state, which consequently indirectly regulates transfer RNA (tRNA) charging to influence protein expression.