Abstract
RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2) protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA). The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site.
Highlights
RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA [1]
In order to express in E. coli mouse Argonaute 2 (mAgo2), its coding sequence [16] was cloned into the expression vector pMAL-c2e in frame with the 3’ end of the DNA encoding the maltose binding protein (MBP) to obtain pMAL-mAgo2 (Figure 1)
Transcription is directed by the Ptac promoter and the encoded protein comprises the polypeptide chain of mAgo2 (859 amino acid residues) fused to the
Summary
RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA (dsRNA) [1]. The crystal structure of full-length Argonaute from Pyrococcus furiosus (PfAgo) has shown that the PIWI domain has an RNase H fold with two catalytic aspartates in positions identical to those found in other. RNase H family enzymes [13] These residues and an adjacent histidine bind a divalent metal ion 2+. Additional insights into the catalytic mechanism of Argonaute proteins have been provided by the determination of the crystal structure of Bacillus halodurans RNase H (Bh-RNase H) in complex with an RNA:DNA hybrid [15]. It was proposed that metal ion B, not observed in the crystal structure of PfAgo, may bind to the active site of Argonautes upon interaction with the substrate [15]. We report the development of an expression system in Escherichia coli for mouse Argonaute 2 (mAgo2) that is suitable for functional and mutational studies
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
