大腸桿菌內CRP對CRISPR/Cas系統, small RNAs及lacI基因之調控功能研究

Abstract

環單磷酸腺苷受體蛋白(cAMP receptor protein; CRP)是大腸桿菌(Escherichia coli)中重要轉錄轉錄因子，CRP被環單磷酸腺苷（cyclic adenosine 5’-monophosphate; cAMP）活化並參與非葡萄糖碳源代謝之基因表現，過去三十年間已知CRP可控制超過400個基因。近年其它重要基因調控系統在大腸桿菌內被發現，例如CRISPR/Cas系統（Clustered regularly interspaced short palindromic repeat/Cas system; CRISPR/Cas system）、小核醣核酸(Small RNAs; sRNAs)可參與基因表現，此外，同一個基因能被兩個不同轉錄調控子控制，構成前饋式迴路(Feed-forward loops; FFLs)，顯示基因調控網路的複雜性。 本研究發現CRP可調控這些不同基因系統，完成大腸桿菌內重要生理功能。近年發現之CRISPR/Cas system，可產生小片段的crRNA(CRISPR-RNA)，來對抗噬菌體感染，被視為細菌的免疫系統。本研究實驗結果證實當培養基添加葡萄糖後，可抑制噬菌體P1在大腸桿菌體內的複製能力，此機制受CRP調控。此外，在大腸桿菌中sRNAs長度介於40-500鹼基，多位於基因非編碼區，目前為止已發現約80個sRNAs，具有多樣的生理功能。本研究發現多達三分之一以上的sRNAs受CRP調控，並可與CRP構成不同型態的FFLs以控制目標基因表現。 在FFLs研究中已知Coherent type 1 FFLs具有加速基因表現至最大量的功能，而Incoherent type 1 FFLs則具有延遲回應環境訊息的功能，顯示這些FFLs對於大腸桿菌在環境中之適應能力具有貢獻。本研究也發現CRP可藉由控制lacI 並與lacZ 形成CRP-LacI-lacZ FFL，此三基因相互之調控關係形成Coherent type 4 FFLs，此調控可能使大腸桿菌在碳源(例如葡萄糖與乳糖)環境中更有效率的進行不同碳源利用。

Cyclic adenosine monophosphate (cAMP) receptor protein (CRP), activated by cAMP in low-glucose environments, is an important transcription factor in Escherichia coli that participates in the gene expression for non-glucose carbon source metabolism. Research in the past 30 years revealed that CRP controls more than 400 genes. Recent discoveries of other important genetic control systems, such as the clustered regularly interspaced short palindromic repeat/Cas system (CRISPRC/Cas system), small ribonucleic acids (sRNAs), and feed-forward loops (FFLs), demonstrated the complexity of the gene regulation network. CRP could accomplish various regulations in E. coli by regulating different genetic control systems. The CRISPRC/Cas system in bacteria could combat phage infection by producing CRISPR–RNA (crRNA). We revealed that the addition of glucose to the medium inhibited the replication capacity of phage P1 in E. coli because of the regulation of crRNA by CRP. sRNAs are important regulatory factors. sRNAs are approximately 40 bp to 500 bp in length and are located in the noncoding region. More than 80 sRNAs with various physiological functions have been reported in E. coli. More than a third of such sRNAs was regulated by CRP, forming FFLs in different forms. The coherent type 1 FFLs could maximize the acceleration of gene expression, and the incoherent type 1 FFLs delayed response to environmental messages. Thus, the formation of FFLs contributed to the adaptability of E. coli to the environment. In addition, CRP repressed lacI gene and formed CRP-LacI-lacZ FFL with lacZ (β-Galactosidase). The interdependent regulatory relationships among these three genes formed the coherent type 4 FFL. Thus, E. coli may use dual carbon (e.g., glucose and lactose) more efficiently during development under different environments.