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| 購買進(jìn)口儀器、試劑和耗材——就在始于2001年的畢特博生物 www.mobfastly.com |
清華大學(xué)生命學(xué)院高寧、雷建林研究組報道了大腸桿菌核糖體大亞基和ObgE復(fù)合物的冷凍電鏡結(jié)構(gòu)，并結(jié)合生化數(shù)據(jù)證明了ObgE與核糖體大亞基結(jié)合的機(jī)制，進(jìn)而探討了ObgE在核糖體大亞基成熟階段、以及氨基酸饑餓條件下對蛋白質(zhì)翻譯的調(diào)控機(jī)理。相關(guān)文章發(fā)表于2014年5月20日的《PLOS biology》雜志上。  圖示為ObgE結(jié)合的50S核糖體亞基復(fù)合物的5.5 Å冷凍電鏡結(jié)構(gòu)及原子模型。 一個正常的細(xì)菌細(xì)胞中包含上萬個由大小兩個亞基組成的核糖體，參與合成細(xì)胞生長所需的各種蛋白質(zhì)。在過去的二十年里，盡管核糖體催化的蛋白翻譯分子過程和機(jī)理得到了精細(xì)的理解，但是蛋白翻譯過程是如何受到細(xì)胞內(nèi)各種的信號通路調(diào)控還不甚明確。當(dāng)生長條件嚴(yán)苛，如氨基酸饑餓的時候，依舊盲目的合成蛋白就顯得得不償失，此時細(xì)菌需要將所有的能量集中于保持存活，細(xì)菌的這種策略叫做“嚴(yán)緊反應(yīng)”(Stringent Response)，其中的氨基酸饑餓反應(yīng)就由一種小分子信號素(p)ppGpp介導(dǎo)。 已有的研究顯示，(p)ppGpp可以抑制一系列細(xì)菌的基本生理過程，包括染色體復(fù)制，基因轉(zhuǎn)錄，最終都間接的影響蛋白質(zhì)翻譯。同時也有一些證據(jù)表明(p)ppGpp也可以直接影響翻譯過程本身，但其中的機(jī)理卻尚不明確。 本研究針對一種細(xì)菌中保守的GTP酶ObgE展開研究。本文首先證實(shí)了ObgE蛋白可以與核糖體大亞基結(jié)合，這一結(jié)合反應(yīng)依賴于核酸，并且在(p)ppGpp結(jié)合的條件下最為緊密。70S核糖體組裝的動力學(xué)研究結(jié)果顯示，ObgE以競爭性的方式結(jié)合于核糖體大亞基上，從而阻礙核糖體小亞基的結(jié)合，從而抑制下游的翻譯起始過程。 冷凍電鏡結(jié)構(gòu)分析表明，ObgE巧妙的結(jié)合于核糖體大小亞基的界面，占據(jù)了眾多翻譯因子的結(jié)合位點(diǎn)，展現(xiàn)出一種典型的 “抗結(jié)合因子”特征。這些結(jié)果表明ObgE作為一種(p)ppGpp結(jié)合蛋白/效應(yīng)因子，可以感知細(xì)胞內(nèi)的生長調(diào)控信號，特異的結(jié)合到處于組裝晚期的不成熟核糖體大亞基中間體和處于翻譯間期的靜息狀態(tài)的成熟大亞基，從而在全局水平下調(diào)細(xì)胞內(nèi)的核糖體組裝及蛋白翻譯過程。 清華大學(xué)生命學(xué)院2010級博士生馮博雅為本文第一作者，清華大學(xué)生命學(xué)院高寧、雷建林、及瑞典烏普薩拉大學(xué)Suparna Sanyal博士為共同通訊作者。清華大學(xué)生命學(xué)院吳嘉煒教授、王志新教授、以及2009級博士生王杰參與了本文中酶活測定的部分。本研究得到了國家基金委自然科學(xué)基金，科技部重大研究計劃，清華大學(xué)自主科研項(xiàng)目的資助，數(shù)據(jù)處理和結(jié)構(gòu)計算得到了國家蛋白質(zhì)科學(xué)基礎(chǔ)設(shè)施(北京)和清華大學(xué)“探索100”高性能計算平臺的支持。 原文摘要： Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase Boya Feng, Chandra Sekhar Mandava, Qiang Guo, Jie Wang, Wei Cao, Ningning Li, Yixiao Zhang, Yanqing Zhang, Zhixin Wang, Jiawei Wu,Suparna Sanyal, Jianlin Lei, Ning Gao Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues. |
購買進(jìn)口儀器、試劑和耗材——就在始于2001年的畢特博生物
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