已知Argonaute蛋白是一種結合RNA的蛋白質,,研究人員已經(jīng)鑒定并分析了酵母Argonaute蛋白的晶體結構。在沉默基因的RNA干擾(RNAi)通路中,,這種Argonaute-RNA復合物起著關鍵作用,,描述真核生物Argonaute蛋白分子結構一直是RNA干擾領域近十年的目標。了解Argonaute晶體結構不僅是理解RNAi生化通路很重要的一步,,也是將來許多實驗的依據(jù),。
在人和其他真核生物中,RNAi通路能通過減少蛋白質的RNA模板來降低細胞內蛋白生產(chǎn),。利用這條通路,,科學家能敲除特定蛋白的表達,從而測定它們在細胞或組織中的作用,;在人類疾病的治療方面,,RNAi通路也相當重要。
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,,用另一條鏈作為向導RNA,,當單鏈RNA匹配上向導RNA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結構,研究人員通力合作,,希望能解決Argonaute結構問題,,結果發(fā)現(xiàn)它是一個具有復雜拓撲結構和許多活動部分的大蛋白,確實是一個令人印象深刻的分子機器,在結構中伴有少量RNA,,這些RNA的摻入將Argonaute蛋白轉變成一種含4元件活化部位的激活態(tài),。這樣,一個長期以來的謎,,即什么構成了"消失的"第四元件,,也就得到了解答。有這個結構在手,,科學家現(xiàn)在就能更好地了解它怎么運作,。(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