被称为RNA干扰(RNA interference, 或RNAi)的现象的首次发现，是在外源的多拷贝转基因被引入牵牛花后所激起的一个特定序列的基因沉默响应中。Argonaute是RNAi效应复合体中的标志组分，本期刊登了测量其晶体结构的研究文章，该文曾在7月29日首次发表在科学特快上。本期封面与此有关，分别刊登了Argonaute的晶体结构图像以及RNA干扰造成的牵牛花的双色彩图案。本期还发表了一篇相关的研究评述。
Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity
Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of "Slicer," the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a "stalk"-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as "Slicer." The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.
Fig. 1. Crystal structure of P. furiosus Argonaute. (A) Stereoview ribbon representation of Argonaute showing the N-terminal domain (blue), the "stalk" (light blue), the PAZ domain (red), the middle domain (green), the PIWI domain (purple), and the interdomain connector (yellow). Active site residues are drawn in stick representation. Disordered loops are drawn as dotted lines. (B) Schematic diagram of the domain borders.
Fig. 2. The PAZ domains of PfAgo and hAgo1 have very similar structures. (A) Stereoview diagram of the superposition of C atoms from the PAZ domain of PfAgo (red) and the PAZ domain of hAgo1 (gray). Dotted lines represent disordered regions. (B) Side chains involved in binding the two-nucleotide 3' overhang are shown in stick representation with PfAgo residues in atom colors (carbon, yellow; oxygen, red; and nitrogen, blue), and hAgo1 residues in green.
Fig. 3. PIWI is an RNase H domain. (A) Ribbon diagrams of the PIWI domain, Escherichia coli RNase HI and Methanococcus jannaschii RNase HII. The three structures are shown in a similar view with the secondary structure elements of the canonical RNase H fold in color. Active site residues are shown in stick representation. (B) This view of the active site residues is rotated 180° about the y axis compared with the view in (A). The Mg2+ ion in RNase HI is shown as a pink sphere. A strong difference electron density (>4.5) found in the active site of PIWI that was assigned as a water molecule is shown as a green sphere. Secondary structural elements of the RNase H fold are colored from red to pink (red, orange, yellow, green, blue, purple, pink) as ordered in the protein sequence.
Fig. 4. A model for siRNA-guided mRNA cleavage by Argonaute. (A) View of the electrostatic surface potential of PfAgo indicating a positively charged groove (blue). The approximate location of the active site is marked by a yellow asterisk. This view is slightly tilted on the horizontal axis compared to the view in Fig. 1. Two of the loops were removed for a better view of the groove. (B) A 3' portion of the siRNA (purple) was placed by superposition of the PAZ domain of the hAgo1-PAZ domain RNA complex on the PAZ domain of PfAgo. The passenger strand of the hAgo1-PAZ complex placed in a similar manner was used to model the mRNA strand (light blue) by extending the RNA two nucleotides at the 5' end, and from the middle of that strand along the binding groove toward the active site in PIWI. The phosphate between nucleotides 11 and 12 from the 5' end of the mRNA falls near the active site residues (red). The view is similar to the view in Fig. 1. (C) Schematic depiction of the model for siRNA-guided mRNA cleavage. The domains are colored as in Fig. 1. The siRNA (yellow) binds with its 3' end in the PAZ cleft and the 5' is predicted to bind near the other end of the cleft. The mRNA (brown) comes in between the N-terminal and PAZ domains and out between the PAZ and middle domain. The active site in the PIWI domain (shown as scissors) cleaves the mRNA opposite the middle of the siRNA guide.
