标题: 大肠杆菌 K12内 CRP 蛋白参与调控转录因子之研究
Identification of CRP-regulated transcription factors in Escherichia coli K12
作者: 魏信文
Wei, Shin-Wen
曾庆平
Tseng, Ching-Ping
生物科技学系
关键字: 转录因子;环单磷酸腺苷;环单磷酸腺苷受体蛋白;基因调控;前馈式基因调控回路;transcription factors;cyclic-AMP;cyclic-AMP receptor protein;gene regulation;feedforward loops
公开日期: 2012
摘要: 环单磷酸腺苷受体蛋白(cAMP receptor protein; CRP)是一种转录因子,在低葡萄糖的环境中它能被cAMP活化而帮助参与非葡萄糖来源代谢的基因表现,因此CRP被认为与代谢抑制(catabolite repression)有关。 在大肠杆菌基因调控网路研究中,CRP已知可调控超过400个基因。CRP也能透过其他转录因子形成前馈式基因调控回路(FFLs)来调节基因表现以适应环境改变。先前FFLs相关研究结果发现coherent type 1 FFLs具有延迟回应环境讯息的功能,而incoherent type 1 FFLs则具有加速基因表现至最大量的功能,显示这些前馈式基因回路的形成对于大肠杆菌在环境中之适应能力具有贡献。 目前CRP所形成的FFLs并没有完整研究资讯,因此本论文结合生物资讯CRP结合序列预测工具以及实验验证找出在大肠杆菌中所有被CRP调控的转录因子,并分析所有可能受CRP调控之FFLs。
在本研究中,CRP序列预测结果显示大肠杆菌190个转录因子中有85个转录因子启动子区至少有1个CRP结合序列,其中37个转录因子已有文献报导受到CRP调控。 另外48个转录因子我们透过EMSA与real-time PCR验证后得知有37个转录因子确实会受到CRP调控,因此受CRP调控的转录因子数增加至74个。整合本研究结果与Ecocyc资料库中基因调控关系纪录,CRP总共调控了750个基因,并形成了153个FFLs。其中特别的是受CRP直接调控的459个基因中有370个 (80%) 可以受CRP调控之转录因子形成FFLs,此发现显示受CRP调控之基因同时会受到环境中其他调控讯息的影响。进一步分析这些转录因子与CRP形成的FFLs,我们发现coherent type I和incoherent type I数目最多,而且功能主要为调控碳源代谢与运输蛋白相关基因。
本研究系统性的找出大肠杆菌基因体中受CRP调控的FFLs,同时也提供了详细的FFL调控资讯。这些资讯,将可进行更深入的研究来揭开CRP在大肠杆菌K12中之调控功能。
The cyclic-AMP receptor protein (CRP), a transcription factor (TFs), mediates catabolite repression and regulates more than 400 genes in Escherichia coli. CRP can regulate other TFs to respond to environmental change through forming feedforward loops (FFLs). FFLs are known to have the ability to respond to environmental signals. For example, coherent type I FFLs have sign-sensitive delay function, and incoherent type I FFLs can accelerate response-time. CRP is highly induced in absence of glucose, such as in the urinary tract and colon. Therefore, CRP may use FFLs to respond to environmental nutrients and enhance the survival ability of E. coli. However, a comprehensive understanding of the regulatory role of CRP in TFs is unclear. In this study, a computational model was applied to predict potential CRP binding sites in the promoter regions of 190 TFs in E. coli (RegulonDB 7.4), and the functions of CRP-regulated FFLs were analyzed.
Results showed that 85 of the TFs contained at least one CRP binding site in the promoter region. These predicted CRP binding sites were confirmed by electrophoretic mobility shift assay, and real-time PCR. A total of 37 novel CRP-regulated TFs were identified. Combining these new results with reported regulatory relationships, we found that 750 genes were direct or indirect-regulated by CRP-regulated TFs, and 153 novel FFLs were generated. Interestingly, 80% (370/459) of CRP direct-regulated genes were also indirect-regulated by CRP, and this finding show that the target gene of CRP was flexibly cooperated by other TFs to respond to environmental signals. Furthermore, coherent type I and incoherent type I were major types in CRP-regulated FFLs. The function analysis of CRP-regulated FFLs shows that those FFLs mainly regulated genes relative to carbon metabolism and transportors.
In conclusion, we used a genome-wide screening approach to expand knowledge on the CRP-regulation network in E. coli K12.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079928509
http://hdl.handle.net/11536/49959
显示于类别:Thesis