The crystal structure of the giardia intact dicer enzyme shows that the PaZ domain, a module that binds the end f dsRNA, is separated from the two catalytic rNase III domains by a flat, positively charged surface The 65 angstrom distance between the paz and RNase Iii domains matches the length spanned by 25 base pairs of rna. thus, Dicer itself is a molecular ruler that recognizes dsRNa and cleaves a specified distance from the helical end Bridging RNase Ill connec RNase Ilib 6 Platform 3 PAZ
17 The crystal structure of the Giardia intact Dicer enzyme shows that the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic RNase III domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end
RISC: the key component is Argonaute(AGO) Microprocessor 5 3 Nucleus Pre-mIRNA mRNA Cytoplasm 份2 Target RNA cleavage o2 P MAGO2 RISC recycling Dicer TRBP Stand selecton RISC and separaton
18 RISC: the key component is Argonaute (AGO) AGO2 Dicer TRBP
Argonaute (AGO): A large protein family that constitutes key components of rIscs AGo proteins are characterized by two unique domains, PAZ and PIWI, whose functions are not fully understood. Current evidence suggests that the paz domain binds the 3-end two -nucleotide overhangs of the siRNA duplex, whereas the PIWi domain of some AGO proteins confers slicer activity. PAZ and PIWI domains are both essential to quide the interaction between the siRNA and the target mRna for cleavage or translational repression Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave the mRNA target, whereas AGo1 and AGo3 do not 19
19 Argonaute (AGO): A large protein family that constitutes key components of RISCs. ---AGO proteins are characterized by two unique domains, PAZ and PIWI, whose functions are not fully understood. Current evidence suggests that the PAZ domain binds the 3’-end two-nucleotide overhangs of the siRNA duplex, whereas the PIWI domain of some AGO proteins confers slicer activity. PAZ and PIWI domains are both essential to guide the interaction between the siRNA and the target mRNA for cleavage or translational repression. ---Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave the mRNA target, whereas AGO1 and AGO3 do not
Crystal Structure of argonaute and its Implications for riSc Slicer Activity 3 SEPTEMBER 2004 VOL 305 SCIENCE Ji-Joon Song, 2 Stephanie K. Smith,2 Gregory JHannon, Mid PIWI Fig. 1. Crystal structure of P. furiosus Argonaute.(A)Stereoview ribbon representation of Argonaut te 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 20
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A model for siRNA-guided mRna cleavage by Argonaute B SiRNA PAZ mRNA Fig. 4. A model for siRNA-guided aRNa cleav (A)View of the electrostatic sur- C face potential of PfAgo indicat- ing a positively charged groove (blue). The approximate location 5 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 re- moved for a better view of the groove.(B)A 3 portion of the siRNA (purple) was placed by su- perposition of the PAz domain of the hAgo 1-paz domain rNa 21 complex on the paz domain of
21 A model for siRNA-guided mRNA cleavage by Argonaute