Related Experiment Video
Updated: Jun 21, 2026
12:24
PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
Published on: July 2, 2010
Binding and cleavage specificities of human Argonaute2
Walt F Lima1, Hongjiang Wu, Josh G Nichols
1Department of Molecular and Structural Biology, Isis Pharmaceuticals, Inc., Carlsbad, California 92008, USA. wlima@isisph.com
The Journal of Biological Chemistry
|July 24, 2009
Summary
Argonaute2 (Ago2) endonuclease binds single-stranded RNA more effectively than double-stranded RNA. Divalent cations like Mn2+ enhance Ago2 activity, and its interaction with antisense RNA involves specific 5' terminal nucleotides.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- Argonaute2 (Ago2) is a key endonuclease in RNA interference, responsible for target mRNA degradation.
- Understanding Ago2's binding and cleavage mechanisms is crucial for deciphering gene silencing pathways.
Purpose of the Study:
- To characterize the binding and cleavage properties of recombinant human Ago2.
- To investigate the influence of RNA structure and divalent cations on Ago2 activity.
- To identify the specific interactions between Ago2 and antisense RNA.
Main Methods:
- Recombinant human Ago2 expression and purification.
- In vitro binding and cleavage assays using various RNA substrates.
- Enzyme kinetics studies with different divalent cations (Mg2+, Mn2+).
- RNase protection assays and analysis of modified antisense RNAs.
- Co-immunoprecipitation of transiently expressed Ago2 from HeLa cells.
Main Results:
- Human Ago2 exhibits higher affinity for single-stranded RNA than double-stranded RNA.
- Ago2's RNase H activity is enhanced by Mn2+ compared to Mg2+.
- The 5'-terminal nucleotide of antisense RNA significantly influences Ago2 binding and cleavage.
- Ago2 interacts with the first 14 nucleotides of the antisense RNA and also with regions outside the hybridization site on sense RNA.
- Ago2 interacts with the human immunodeficiency virus type 1 trans-activating response RNA-binding protein (TRBP) via its PIWI domain.
Conclusions:
- Human Ago2 preferentially binds and cleaves single-stranded RNA, with specific requirements for the 5' end.
- Divalent cations, particularly Mn2+, play a critical role in modulating Ago2's enzymatic activity.
- Ago2's interaction with RNA involves both the antisense strand and extended regions of the sense strand.
- Ago2 forms a complex with TRBP, suggesting a role for this interaction in the RNA-induced silencing complex.
Related Concept Videos
02:26Conservation of Protein Domains Over Different Proteins
Protein domains are small structurally independent units that are part of a single amino acid chain. Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
02:23RNA Editing
RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
02:47Ribozymes
The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Ribozymes can be...
01:49Conserved Binding Sites
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
00:49Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
02:28Leaky Scanning
During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA. Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...