大腸桿菌CRP 蛋白調控topA 基因之研究

Abstract

本研究的主要目的是要探究環磷酸腺苷AMP受體蛋白（CRP）在大腸桿菌中對於topA基因扮演了甚麼樣的調控腳色。在真核和原核生物中topA基因表現拓譜異構酶 topoisomerase I來緩解染色體DNA過度緊繃的超螺旋結構。先前的研究顯示topA基因會受到FIS蛋白調控，為了避免CRP突變株CRP調控fis基因的差異影響對topA基因的實驗觀察，我們構築了topA 啟動子區CRP的結合序列的突變株(topA*)。實驗上我們做了電泳遷移率變動分(electrophoretic mobility shift assay, EMSA)及足跡法(footprinting)來驗證topA基因啟動子區的CRP序列，也用即時定量real-time PCR和 lacZ reporter fusion的技術，來驗證CRP對topA基因的調控關係。結果證實CRP蛋白在轉錄層次中會活化topA基因的表現。微陣列分析結果也證實topA受到CRP蛋白的活化。 此外，為了證實CRP對topA基因的調控也具有生理上的影響，我們比較了大腸桿菌野生型和突變株中DNA的超螺旋結構差異。實驗以測量兩株菌中DNA的表面電漿薄膜共振來觀察超螺旋結構的差異，該方法避免了以往使用有毒的chloroquine來進行電泳分析，最終我們發現有177個基因受到CRP調控topA基因影響，也證實了CRP可藉由調控topA基因影響大腸桿菌的DNA超螺旋結構。

The present study aimed to investigate the effect of the regulatory role of cyclic adenosine monophosphate (cAMP) receptor protein (CRP) in the regulation of topA gene expression in Escherichia coli. topA gene codes for topoisomerase I and is responsible for relaxing the negative supercoils in the chromosomal DNA of prokaryotic and eukaryotic cells. We provided an experimental evidence to indicate that CRP regulated the topA gene by real-time PCR, lacZ reporter fusion, electrophoretic mobility shift assay and DNaseI foot-printing assay. We also determined the DNA supercoils to explore the regulation effect of CRP on the topA gene using surface plasmon resonance (SPR) assay. To understand the effect of CRP-regulated topA on DNA supercoiling, we compared the DNA supercoils of wild type with the CRP binding site mutant strain. Previous studies showed that gyrA is regulated by CRP, and the expression of gyrA gene will change the DNA supercoiling when E. coli is under high energy state or high growth rate. To avoid these indirect effects and to clarify the direct effect of CRP control on the topA gene expression, we performed a strategy that constricted the point mutation strain topA* by counter selection method. The CRP-binding sequence of topA promoter region on topA* chromosome was changed to no CRP binding ability sequence. Therefore, we could determine the DNA supercoiling changes only by the CRP regulating topA gene expression of the wild type and topA* strain by surface plasmon resonance assay to object the changes. In conclusion, we verified the CPR-binding site of topA gene and found that CRP was an activator to topA gene. Results showed that CRP-regulated topA gene affected the DNA supercoils and transcription. There are 178 genes changing expression distinctly when DNA supercoiling alters. Hence, CRP plays an important role in regulating topA gene expression in E. coli.