大腸桿菌 K12內 CRP 蛋白參與調控轉錄因子之研究

Abstract

環單磷酸腺苷受體蛋白(cAMP receptor protein; CRP)是一種轉錄因子，在低葡萄糖的環境中它能被cAMP活化而幫助參與非葡萄糖來源代謝的基因表現，因此CRP被認為與代謝抑制(catabolite repression)有關。 在大腸桿菌基因調控網路研究中，CRP已知可調控超過400個基因。CRP也能透過其他轉錄因子形成前饋式基因調控迴路(FFLs)來調節基因表現以適應環境改變。先前FFLs相關研究結果發現coherent type 1 FFLs具有延遲回應環境訊息的功能，而incoherent type 1 FFLs則具有加速基因表現至最大量的功能，顯示這些前饋式基因迴路的形成對於大腸桿菌在環境中之適應能力具有貢獻。 目前CRP所形成的FFLs並沒有完整研究資訊，因此本論文結合生物資訊CRP結合序列預測工具以及實驗驗證找出在大腸桿菌中所有被CRP調控的轉錄因子，並分析所有可能受CRP調控之FFLs。 在本研究中，CRP序列預測結果顯示大腸桿菌190個轉錄因子中有85個轉錄因子啟動子區至少有1個CRP結合序列，其中37個轉錄因子已有文獻報導受到CRP調控。 另外48個轉錄因子我們透過EMSA與real-time PCR驗證後得知有37個轉錄因子確實會受到CRP調控，因此受CRP調控的轉錄因子數增加至74個。整合本研究結果與Ecocyc資料庫中基因調控關係紀錄，CRP總共調控了750個基因，並形成了153個FFLs。其中特別的是受CRP直接調控的459個基因中有370個 (80%) 可以受CRP調控之轉錄因子形成FFLs，此發現顯示受CRP調控之基因同時會受到環境中其他調控訊息的影響。進一步分析這些轉錄因子與CRP形成的FFLs，我們發現coherent type I和incoherent type I數目最多，而且功能主要為調控碳源代謝與運輸蛋白相關基因。 本研究系統性的找出大腸桿菌基因體中受CRP調控的FFLs，同時也提供了詳細的FFL調控資訊。這些資訊，將可進行更深入的研究來揭開CRP在大腸桿菌K12中之調控功能。

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.