大腸桿菌內 RNase E 和 poly (A) 聚合酶對延胡索酸酶 A mRNA 降解機制之調控

Abstract

中文摘要 在大腸桿菌內，延胡索酸酶（fumarase）是檸檬酸循環（TCA cycle）中將延胡索酸（fumarate）與蘋果酸（L-malate）相互轉換的重要酵素。根據我們先前研究結果發現，延胡索酸酶 A（Fumarase A）的mRNA穩定性（stability）會隨著細菌生長速率上升而增加，此結果顯示影響fum A的mRNA穩定性受細菌生長速率所影響。 近年來在大腸桿菌內有兩個重要發現，其一為mRNA的分解酶是以複合體的形式存在，稱之為degradosome：分別由RNase E、polynucleotide phosphorylase (PNPase)、RNA helicase (RhlB)、polyphosphate kinase (PPK)以及enolase所組成。另一個是poly (A) 聚合酶，它可以合成poly (A) tail連接於mRNA的3’端，其作用在增加mRNA的降解速率。由批次培養的結果得知，RNase E缺失後，fum A mRNA的穩定性明顯提高，並生成一段中間降解片段產物，證實RNase E為主導 fum A的mRNA降解機制的核糖核酸分解酶。而poly (A) 聚合酶缺失後，fum A的mRNA穩定性並沒有改變，但是在RNase E與poly (A) 聚合酶同時缺失時，中間降解片段產物消失，而fumA mRNA的穩定性比RNase E單獨缺失的情況下還要高。據此推測poly (A) 聚合酶可能藉由調控一個未知的核糖核酸內切酶的活性來參與其降解機制。 在16S rRNA方面，我們首次證實RNase E與poly (A) 聚合酶同時缺失會干擾16S rRNA的生成作用，但至目前為止並未見到其它相關之研究。 由連續式培養我們發現RNase E缺失後，fum A mRNA的半衰期延長2 ~ 3倍，但是其穩定性受生長速率所影響的現象依舊存在。而根據文獻指出， hfq為導致ompA mRNA的穩定性受生長速率所影響之調控因子，但是由我們的實驗暗示hfq可能對 fum A mRNA的穩定性沒有影響。

Abstract Fumarases is an importance enzyme to catalyze the Interco version of fumarate to L-malate in Escherichia coli. Early studies have shown that the tricarboxylic acid cycle gene, fum A mRNA stability increased with cell growth rate. The mechanism of mRNA degradation in E.coli have been investigated extensively during the last decade. One of the important developments has been recently discovered that the degradosome is a mutilprotein complex containing an endoribonuclease (RNase E), an exoribonuclease (polynucleotide phosphorylase), a DEAD box helicase (RhlB), a polyphosphate kinase (PPK), an enolase, and that oligo (A) tails synthesized by poly (A) polymerase facilitate the degradation of mRNAs have been discovered. In this study the stability of fum A mRNA in the rne single mutant was higher than in wild-type strain, and the mRNA decay intermediate fragment has been found. These results indicated that RNase E was the key ribonuclease for fum A mRNA degradation. Although there was no significant effect on fum A mRNA stability in the pcnB single mutant, the inactivation of rne and pcnB genes significantly increased the stability of fum A mRNA. Therefore, we suppose that poly (A) polymerase may involve in the fum A mRNA degradation by some unknown endoribonucleases activity. The RNase E, RnaseG and RNase III have been shown to be involved into the maturation of 16S rRNA. Interestingly, the results of this study suggested that poly (A) polymerase also related to the processing of 16S rRNA. The half-life of ompA mRNA increase with growth rate in wild-type strain but growth rate-dependent regulation of ompA mRNA is abolished in hfq- strain ; however, in our study, the stability of fum A mRNA did not change in hfq- strain.