标题: | 大肠杆菌CRP 蛋白调控topA 基因之研究 Regulatory role of cAMP Receptor Protein (CRP) on topA gene in Escherichia coli |
作者: | 蔡瀚霆 Tsai, Han-Ting 曾庆平 Tseng, Ching-Ping 分子医学与生物工程研究所 |
关键字: | 环磷酸腺苷AMP受体蛋白;topA 基因;DNA 超螺旋结构;CRP;topA;DNA supercoils |
公开日期: | 2015 |
摘要: | 本研究的主要目的是要探究环磷酸腺苷AMP受体蛋白(CRP)在大肠杆菌中对于topA基因扮演了甚么样的调控脚色。在真核和原核生物中topA基因表现拓谱异构酶 topoisomerase I来缓解染色体DNA过度紧绷的超螺旋结构。先前的研究显示topA基因会受到FIS蛋白调控,为了避免CRP突变株CRP调控fis基因的差异影响对topA基因的实验观察,我们构筑了topA 启动子区CRP的结合序列的突变株(topA*)。实验上我们做了电泳迁移率变动分(electrophoretic mobility shift assay, EMSA)及足迹法(footprinting)来验证topA基因启动子区的CRP序列,也用即时定量real-time PCR和 lacZ reporter fusion的技术,来验证CRP对topA基因的调控关系。结果证实CRP蛋白在转录层次中会活化topA基因的表现。微阵列分析结果也证实topA受到CRP蛋白的活化。 此外,为了证实CRP对topA基因的调控也具有生理上的影响,我们比较了大肠杆菌野生型和突变株中DNA的超螺旋结构差异。实验以测量两株菌中DNA的表面电浆薄膜共振来观察超螺旋结构的差异,该方法避免了以往使用有毒的chloroquine来进行电泳分析,最终我们发现有177个基因受到CRP调控topA基因影响,也证实了CRP可藉由调控topA基因影响大肠杆菌的DNA超螺旋结构。 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. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT070157112 http://hdl.handle.net/11536/125686 |
显示于类别: | Thesis |