已知Argonaute蛋白是一種結(jié)合RNA的蛋白質(zhì),,研究人員已經(jīng)鑒定并分析了酵母Argonaute蛋白的晶體結(jié)構(gòu)。在沉默基因的RNA干擾(RNAi)通路中,,這種Argonaute-RNA復(fù)合物起著關(guān)鍵作用,,描述真核生物Argonaute蛋白分子結(jié)構(gòu)一直是RNA干擾領(lǐng)域近十年的目標(biāo)。了解Argonaute晶體結(jié)構(gòu)不僅是理解RNAi生化通路很重要的一步,,也是將來許多實(shí)驗(yàn)的依據(jù),。
在人和其他真核生物中,RNAi通路能通過減少蛋白質(zhì)的RNA模板來降低細(xì)胞內(nèi)蛋白生產(chǎn),。利用這條通路,,科學(xué)家能敲除特定蛋白的表達(dá),從而測定它們在細(xì)胞或組織中的作用,;在人類疾病的治療方面,,RNAi通路也相當(dāng)重要。
RNAi依賴兩種蛋白,,即Dicer 和Argonaute,,其中Dicer識別雙鏈RNA(dsRNA),鎖定在dsRNA上,,并將其切成21-23個核苷酸長的碎片,;Argonaute識別上述的dsRNA碎片,清除雙鏈RNA中一條鏈,,用另一條鏈作為向?qū)NA,,當(dāng)單鏈RNA匹配上向?qū)NA序列時,Argonaute即裂解此單鏈RNA,,從而防止它作為蛋白生產(chǎn)的模板,。
為了測定Argonaute結(jié)構(gòu),研究人員通力合作,,希望能解決Argonaute結(jié)構(gòu)問題,,結(jié)果發(fā)現(xiàn)它是一個具有復(fù)雜拓?fù)浣Y(jié)構(gòu)和許多活動部分的大蛋白,確實(shí)是一個令人印象深刻的分子機(jī)器,,在結(jié)構(gòu)中伴有少量RNA,,這些RNA的摻入將Argonaute蛋白轉(zhuǎn)變成一種含4元件活化部位的激活態(tài)。這樣,,一個長期以來的謎,,即什么構(gòu)成了"消失的"第四元件,也就得到了解答,。有這個結(jié)構(gòu)在手,,科學(xué)家現(xiàn)在就能更好地了解它怎么運(yùn)作。(生物谷bioon.com)
doi:10.1038/nature11211
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Structure of yeast Argonaute with guide RNA
Kotaro Nakanishi, David E. Weinberg, David P. Bartel, Dinshaw J. Patel
The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2?? crystal structure ofKluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage