大腸桿菌中核醣核酸E之N端及C端在細胞膜結合及細胞分裂調控機制之研究

在大腸桿菌中，核醣核酸E (RNase E) 是組成RNA降解體(RNA degradosome) 的核心蛋白，且其在RNA修飾及降解的過程中扮演著重要的角色。我們實驗室早期的研究首度發現並提出，RNase E組成的RNA降解體可透過其N端結合至細胞膜(1-602 aa)。接著，近期研究證明RNase E可以藉由其高度保留的N端(1-499 aa)所組成的帶正電表面與酸性脂質結合。此細胞膜結合可穩定蛋白質的結構及增加與受質結合的能力以加強其酵素催化能力。在本研究中，我們開發並應用了一套適合用來研究RNase E於活體細胞內分佈的螢光成像技術。根據Arai等人的研究(2001)，在兩個蛋白質中間加入一段α-helix序列，可以有效的分開兩個蛋白質並維持其正常功能。因此我們於表達載體上表現RNase E，並於其C端接上mWasabi螢光蛋白，再加入一段α-helix序列來分開這兩個蛋白質以維持蛋白質的正常功能來避免蛋白質過度集結。另外，此組合蛋白的合成可受阿拉伯糖誘導。最後，再將此表達載體送入KSL2010*(原生rne剔除)菌株中，以避免原生RNase E的干擾。此方法避免了原生蛋白質的干擾以及蛋白質過度集結的影響，因此可以提供絕佳的螢光蛋白解析度以利我們後續的研究。本研究的結果佐證了先前生化分析的結果(Murashko et al., 2012)， RNase E的N端及C端皆具有各自獨立的細胞膜結合位。接著，我們更進一 1 步的使用連續記錄成像的方式，清楚的指出了全長RNase E及各個N端子域 (RNase H 或 S1或5’sensor 或 DNase I)剔除的蛋白在初期階段會先聚集於細胞兩極接著再移動至其他細胞膜區域。而單獨N端或C端的RNase E蛋白則聚集於靠近細胞膜的區域接著再移動至整個細胞中。本研究中建立的這套影像系統，將有利於我們後續探討RNase E及其他RNA降解體組成蛋白之間的交互作用。接著，我們使用MetaMorph軟體，以影像為基準來計算細胞大小的結果指出，若剃除RNase H、S1或DNase I子域，會導致細胞變長。所以我們推論，這些子域可能是維持RNase E酵素活性的重要子域，才會進而影響細胞分裂的調控能力。而這些子域對RNase E酵素活性的重要性，仍需進一步的生化分析才能確定。最後，根據我們的結果，我們提出了一個假設。在大腸桿菌中，有一個未知的細胞分裂抑制因子”X factor” 蛋白，而此”X factor”的表現量受到RNase E的調控，因此RNase E的活性才會進而影響細胞分裂。

關鍵字

大腸桿菌

並列摘要

In Escherichia coli (E. coli), RNase E is a core protein of the RNA degradosome that plays a major role in RNA processing and mRNA decay. Previously our lab’s finding showed that RNase E-based degradosome can tether to the cytoplasmic membrane by the N-terminal region of RNase E (1-602 aa). Recent study from our lab has shown that the reconstituted highly conserved N-terminal fragment (NRne, residues 1-499) of RNase E, specifically binds to anionic phospholipids of the membrane through electrostatic interactions. This binding of NRne to the membrane induces changes in protein secondary structure and stabilizes the protein-folding state. Such changes in protein structure and stability, enhances the binding affinity of RNase E to its substrates and increases enzymatic activation. In the present study, first, we have developed an approach for subcellular localization of RNase E polypeptides using fluorescence imaging system. According to the study of Arai et al. (2001), an α-helix linker effectively separated two functional proteins and maintained their function. Therefore, we fused a fluorescent protein mWasabi to C-terminal of RNase E through an α-helix linker which separated these two proteins and minimized protein aggregation. The chimeric polypeptide under the control of an arabinose-induced promoter was then ectopically expressed by a plasmid in endogenous rne deleted KSL2010* strain. The developed method 3 prevented the influence of endogenous RNase E and minimized aggregation of proteins, which resulted in better resolution for subcellular localization. Our results confirmed that both N- and C-terminal parts of RNase E have independent membrane binding sites, which is consistent with our previous biochemical finding (Murashko et al., 2012). Furthermore, the time-lapse images demonstrated that after addition of inducer, full-length RNase E and the N-terminal subdomain (RNase H or S1 or 5’ sensor or DNase I) deleted constructs were initially localized to the poles and then spread onto whole membrane regions. Moreover, the N- or C-terminal of RNase E alone was initially localized at the periphery region and/or cell poles and subsequently dispersed within whole cell. The present imaging system can be used for further investigation for in vivo mapping of interaction between RNase E and its degradosome component proteins. Finally, the analysis of NRne subdomain deletions by image-based cells size calculation (by using software, MetaMorph) showed that RNase H, S1 and DNase I domains had a significant effect on cell size. These results suggest that these domains might play a key role in the enzymatic activity of RNase E which consequently affects cell division, but this needs to be confirmed by biochemical assays. Collectively, according to our results, we propose that mRNA levels of an unknown “X-factor” protein, assumed to be a cell division regulator, might be controlled by RNase E, thereby influencing cell division.