| Summary: | 碩士 === 國立交通大學 === 分子醫學與生物工程研究所 === 103 === 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.
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