cAMP對大腸桿菌ompA mRNA穩定性之研究

Abstract

大腸桿菌之small RNA(sRNA)藉由序列互補的方式，與Hfq協同結合到messenger RNA上，可調控messenger RNAs穩定性與轉譯。先前研究指出cAMP-CRP活化一些sRNA基因表現，例如Spot42與RyeE，並藉由結合到sRNA promoter區域來調控。cAMP-CRP在大腸桿菌又扮演transcriptional activator角色，藉由結合到promoter，增加與RNA polymerase之間的交互作用，活化基因的表現。 本研究將大腸桿菌野生菌株與cya基因突變菌株，分別培養在LB與LB加glucose培養液，結果發現當glucose存在或cya基因突變下，ompA mRNA的表現量明顯下降，結果顯示cAMP可調控ompA基因的表現，我們更進一步分析ompA mRNA在early log-phase與log-phase的穩定性，發現當cAMP不存在時，ompA mRNA穩定性下降快速，結果說明了cAMP可增加ompA mRNA的穩定性。 迄今cAMP影響mRNA穩定性的機制並不清楚，我們推測ompA mRNA在post transcription層次上的穩定性並非藉由cAMP直接的影響。根據northern實驗分析野生菌株與cya突變菌株，結果顯示hfq基因表現受cAMP所影響。但並未找到會影響ompA mRNA穩定性並且是藉由cAMP調控的sRNA，因此我們認為Hfq可能是參與調控ompA mRNA穩定性機制中的主要者。 先前研究已證實ompA mRNA穩定性會受Hfq所降解，由於我們發現cAMP的存在確實降低了hfq mRNA的量。因此推測增加ompA mRNA穩定性是藉由降低hfq基因的表現所造成。

In E. coli, small RNAs primarily serve as regulators with Hfq to modulate messenger RNA stability and translation efficiency by base-pairing with target messenger RNAs. Previous studies have reported that cAMP-CRP regulates small RNA expression by modifying promoter activity, such as spot42 and ryeE gene. As far as we know, the cAMP-CRP acts as a transcriptional activator in E. coli, by binding to promoters and enhancing the interaction with RNA polymerase. In this research, E. coli wild-type and cya mutant strains were grown in LB and LB+Glc, respectively. We found that the amount of ompA mRNA was down-regulated when glucose was added or cya was mutated. These results suggested that cAMP played a regulatory role on ompA expression. Furthermore, we analyzed ompA mRNA stability in early log-phase and log-phase. When cAMP was absent, the ompA mRNA was degraded rapidly. This result indicated that the stability of ompA mRNA was elevated by cAMP. So far, the medchanism of cAMP regulation of RNA stability is not clear. In this study, we propose that post-transcription of ompA mRNA was not directly affected by cAMP. According to the northern blot analysis of wild type and cya mutant, the results showed that the expression of hfq encode RNA chaperone was affected by cya. However, sRNAs were not found to involve in ompA mRNA stability. Thus we suggested that Hfq may participate in regulation of ompA mRNA stability. The Hfq down-regulates ompA mRNA stability which has been demonstrated in previous studies. In this study, we found that the amount of hfq mRNA was down-regulated while cAMP was present. Therefore, the stability of ompA mRNA may be increased by reducing hfq gene expression.