大腸桿菌內環腺苷磷酸受體蛋白(CRP)之前饋式迴路(FFL)基因調控研究

Abstract

環腺苷磷酸受體蛋白(cAMP receptor protein, CRP)是大腸桿菌(Escherichia coli, E. coli )中重要的轉錄因子(transcription factor, TF)，在轉錄層次上CRP可以調控超過400個基因。當葡萄糖濃度低於0.3 mM的環境下，CRP和環腺苷酸(cyclic adenosine 5′- monophosphate, cAMP)會形成複合體。此複合體結合在去氧核醣核酸(deoxyribonucleic acid, DNA)的保守序列上，調控基因表現。前饋式迴路(feed-forward loop, FFL)是一種基因調控模式，由兩個TFs及一個目標基因所組成，FFL可以分為八種型態。到目前為止，在E. coli中被證實存在調控一致型1(coherent type 1, C1)FFL和調控不一致型1(incoherent type 1, I1)FFL。本研究目的發現環腺苷磷酸受體蛋白新的前饋式迴路(CRP-FFLs)。我們結合微陣列數據、生物資訊方法及資料庫找出新的CRP-FFLs，再利用電泳遷移分析(electrophoretic mobility shift assay, EMSA) 與即時定量聚合酶連鎖反應(quantitative polymerase chain reaction, Q-PCR)證實88個基因會受CRP調控。經資料庫重新整理後有56個TFs及483個目標基因可以形成585個CRP-FFLs，結果我們發現八型的CRP-FFLs、多重內部二次輸入節點的CRP-FFLs(multi-Y CRP-FFL)及多重輸出節點的CRP-FFLs(multi-output CRP-FFL)基因調控方式都存在於E. coli中。此外，藉由基因功能體學(gene ontology, GO) 分析CRP-FFLs在E. coli中扮演的生理功能，發現coherent type 1 CRP-FFLs、coherent type 4 CRP-FFLs與 incoherent type 1 CRP-FFLs主要參與碳水化合物的運輸。而coherent type 2 CRP-FFLs與 incoherent type 3 CRP-FFLs主要參與氮的代謝。最後incoherent type 4 CRP-FFLs則是參與電子傳遞鏈。我們接著建立八型突變株，添加不同濃度的cAMP，證實八型CRP-FFLs存在於E. coli中。這個研究不但擴充E. coli中CRP-FFLs的數目、也了解八型CRP-FFLs在E. coli中所扮演的生理功能，並發現在E. coli中受CRP調控的基因中有半數的基因是藉由FFL的形式被調控。

Cyclic AMP receptor protein (CRP) is an important transcription factors (TF) in Escherichia coli, it can regulate about 400 genes in transcriptional level. When glucose concentrations less than 0.3 mM in the medium, CRP can form cAMP-CRP complex. It will bind to conserved DNA sequence, and affects genes expression. Feed-forward loop (FFL) is one of a network motif composed of two TFs and one target gene. FFLs can be divided into eight types. Until now, the physiological functions were confirmed on coherent type 1 and incoherent type 1 in E. coli. The aim of this studt is to investigate the novel CRP mediated FFLs in E. coli. Here, we combines microarray data, bioinformatics method and database to find novel CRP mediated FFLs. The 88 genes directly regulated by CRP were validated by electrophoretic mobility shift assay (EMSA) and quantitative polymerase chain reaction (Q-PCR). The results showed that 56 TFs and 483 genes could form 585 CRP-FFLs in E. coli. We found that eight types of CRP-FFLs, multi-Y CRP-FFL and multi-output CRP-FFL in all existed in E. coli. In addition, We identify the physiological role of CRP-FFLs by Gene ontology (GO) analysis. We find that coherent type 1 CRP-FFLs, coherent type 4 CRP-FFLs and incoherent type 1 CRP-FFLs are related to carbohydrate transport. The coherent type 2 CRP-FFLs and incoherent type 3 CRP-FFLs are related to nitrogen metabolism. The incoherent type 4 CRP-FFLs are related to aerobic electron transport chain. Furthermore, we construct different mutant strains and add various concentration of cAMP to verify that eight types CRP-FFLs exist in E. coli. Finally, this work not only expand the number of CRP-FFLs and realize the biological function of eight types of CRP-FFLs in E. coli, but also found that half of genes regulated by CRP to form FFLs in E. coli.