cAMP-CRP對大腸桿菌 ompA 與 fumA、fumC 基因在轉錄與轉錄後調控之研究

Abstract

Cyclic AMP (cAMP)與 cAMP receptor protein (Crp) 在大腸桿菌扮演重要轉錄調控子(transcription factor)，會調控細胞內數百個基因的轉錄 (transcription)作用。過去研究大腸桿菌mRNA降解機制以outer membrane protein A (ompA) 之mRNA降解為模型，在我們的研究中發現ompA 的mRNA穩定性會因為葡萄糖加入而降低，由於葡萄糖加入會降低細胞內cAMP含量，使用Δcya (cAMP缺失株)突變株也驗證出cAMP會正向調控ompA mRNA的穩定性。已知Host factor I (Hfq)蛋白質、ribonuclease E與small RNA中的MicA 三者會影響ompA 的mRNA穩定性，以real-time PCR方法發現hfq會被cAMP調控，我們進一步利用Northern blot analyses、Western blot analysis 方法，證實在Δcya 突變株中Host factor I (Hfq)表現量會比野生株多，再透過Electrophoretic mobility shift assays (EMSA)發現cAMP-Crp會結合hfq的啟動子(promoter)上游-183 與 -146位置，將含有這一結合位置的hfq 啟動子構築至lacZ fusion，也證實了cAMP-Crp以轉錄調控方式負向調控hfq表現量。為驗證上述的結果及cAMP是如何經由Hfq調控ompA mRNA降解，我們使用會受cAMP正向、負向及無調控的三種不同啟動子構築至hfq基因前，利用這些在相同cAMP情況下卻能有上升、下降、及不變的Hfq表現量，來觀察ompA mRNA的半衰期，結果證實了cAMP-Crp會透過Hfq間接正向調控ompA 的mRNA穩定性，同樣的結果在會受Hfq影響降解的fur mRNA也改變降解速率，因此我們推論cAMP可間接調控mRNA降解是普遍存在於會受Hfq調控穩定性的mRNA上。因此我們證實了cAMP-Crp會作用在轉錄後層面，是經由轉錄調控hfq表現量來達成。

接著我們利用連續式培養方式，由於TCA cycle中之各fumarase基因受氧氣含量影響，在不同含氧量下，測量各fumarase (fum) 基因的lacZ fusion表現量，結果證實其表現會受到cAMP與Crp調控，進一步以Northern blot analyses驗證fumarase中的fumA在轉錄與轉錄後層面都會被cAMP調控，因為大腸桿菌在連續式培養下不同生長速率會有不同的cAMP表現量，因此我們觀察到fumA基因在不同生長速率下，在轉錄層面會隨生長速率的上升而下降，相反的，發現fumA 之mRNA穩定性會隨生長速率的上升而上升，但FumA蛋白質的表現在不同生長速率會維持穩定不變，此結果是反應了fumA在轉錄與轉錄後相反的調控機制。透過我們的研究，可以了解cAMP-Crp除了會調控基因轉錄，也會間接的調控基因在轉錄後的穩定性，並且利用fumA基因，建立在cAMP-Crp調控下，由DNA到mRNA量與穩定性會使最終蛋白質表現量達到互相協調平衡的模式。

E. coli has developed pleiotropic regulator for gene expression, such as cyclic AMP (cAMP)-cAMP receptor protein (Crp) complex. Glucose is generally regarded as a carbon source to modulate cyclic AMP (cAMP) regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose was present in the LB medium. This effect was associated with the low level of cAMP induced by the glucose. The evidence was further confirmed in an E. coli mutant lacking of cAMP. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were down-regulated in the presence of cAMP, in which we showed that complex formation between cAMP receptor protein (Crp) and cAMP plays a role in binding to a specific promoter region of hfq (between -112 and -87). The finding was substantiated in vivo by an hfq-deficent mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that the repression of hfq expression by cAMP leaded to a cascade regulation of cAMP and Hfq on the stability of ompA mRNA. Additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to others post-transcriptional regulations in E. coli. The regulation of fumarase (fumA, B, C) genes was dependent on growth rate in transcription and post-transcription. Our results showed that cAMP and Crp were an activator for fumA and fumC genes expressions under various oxygen using fumA–lacZ and fumC–lacZ fusion strains. Further experiments also showed glucose catabolite repression on fumA and fumC genes, but no effect on fumB gene. Subsequently, we examined the effect of fumA expression in transcription, post-transcription and translation level by growth rate regulation. In continuous culture, the amount of the fumA mRNA was reduced with increasing growth rate. The fumA mRNA was more stable when growth rate was increased. The contrary results between amount and stability of fumA mRNA caused the same amount of FumA protein with different growth rates. This finding may provide a reference value for the future industrial application for the preparation of recombinant proteins.