Scott Watters '14 and Stephen
Introduction and General Structure
silencing is one
for post transcriptional
regulation of RNA within eukaryotic cells. Unique long double
RNA’s are processed into microRNA
(miRNA) which are then uptaken by the RNA induced silencing
Specifically, miRNA is directly bound by a
(RNP) of the Argonaute (Ago) protein family
(Meister et al, 2004). Once the complementary RNA is bound,
Argonaute proteins either slice the RNA directly, or recruit
additional factors to silence the RNA.
members of the
Argonaute family have conserved PIWI
domains. The PIWI
a structure very
similar to that of the RNase H from bacteria
that this domain is
responsible for the endonucleolytic activity of RISC (Shirle &
a number of hydrophobic and positively charged residues which
account for both Ago affinity for RNA and it’s binding to the
siRNA and miRNA processing protein Dicer (Filipowicz, 2005).
humans, there are 4 different Ago proteins. Of these, only Argonaute 2
has been shown to have mRNA silencing activity (Filipowicz 2005). This
protein has two domains in addition to PIWI
these are N
Ago2 also contains an N-terminal domain as well as
domains which connect PAZ
to the N and
Argonaute2 appears to hold seven nucleotides of the guide RNA in a
this binding is
stabilized by hydrogen bonds,
contacts to the phosphate backbone of the RNA, and Van der Waals
contacts with the sugar bases.
of the stabilizing contacts are through the MID and the PIWI
domains. To be specific, Lysine 566 and Arginine
792 make an ion-dipole interaction with the phosphate on the backbone
of the guide RNA, while Tyrosine 790 makes an H-bond to a phosphate
Tyrosine 804, Serine
Histidine 753 additionally stabilize the phosphates in the RNA
minor contacts, include protein RNA interaction
stabilization of this conformation with the 5’ RNA base
stacking with Tyrosine 529
5’ phosphate of
this base forms H-bonds with Tyrosine 529, Lysine 533, Glutamine
and Lysine 566
All of these amino
acids are located in the MID domain.
of these contacts are base specific, as you would expect for the
protein to be able to bind siRNA of many different sequences.
RNA Specificity and Catalytic Activity
target vs. DNA target
2’ hydroxyl of nucleotide 5 bonds to the amide on the
backbone of Isoleucine 756
hydroxyl of nucleotide
bonds to the
backbone carbonyl of Alanine 221
Additionally, the backbone
carbonyls of Asparagine 562 and
make water mediated contacts to the 2’ hydroxyl of nucleotide
2 (bonds not shown due to lack of consistent water position
data in crystal structure).
this amounts to very little
additionally stabilization provided by the 2’ hydroxyl of the
bases and 2’ fluoro
substitutions do not prevent the binding of siRNAs to Argonaute2.
Argonaute2, the RNA nucleotides have the
Watson-Crick faces (the edges of the nitrogenous bases farthest from
exterior environment in an A-form conformation.
However, Isoleucine 365 is
inserted between bases 6 and 7
introduces a kink in the near A-form structure
the minor-grove edge of
nucleotide 7 is further stabilized in this kinked position by
Methionine 364 and Isoleucine
located on alpha helix 7 on the L2 region of Argonaute2.
nucleotide 7 to another nucleotide possibly
shifts helix 7
thus releasing the kink and allowing nucleotides 6 and
base pair. It
is hypothesized that these
are the reasons why effective pairing to nucleotide 7 is so crucial for
targeting, and it is also thought that the protein could introduce a
the RNA has been sliced to allow it to dissociate from the protein.
PIWI domain of argonaute2 contains the endonucleolytic active site of
the molecule. As with the ribonucleases that PIWI shares homology with,
three carboxylate residues are responsible for this catalytic activity;
groups of D641, D669, and either E683 or E673 (Rivas et al,
residues coordinate with one of two Mg2+ ions which
catalyze the hydrolysis of the 3’ phosphodiester bond of RNA
(bonds to metal ions not shown as protein was not crystalized with
PIWI domain contains many of the residues responsible
for argonaute’s protein-protein binding. A number of
aliphatic amino acids
engage in hydrophobic interactions with GW proteins (so called for
increased quantity of glycine and tryptophan residues). Two separate
hydrophobic pockets interacts with tryptophan residues of an associate
protein. In the first L650, I651, Y654, K660, L694, and Y698 pack
against an inserted tryptophan
In the second, the side chains of F587, P590, V591, A620, F653, and
F659 stack against a tryptophan
while the main chain carbonyl of
F587 hydrogen bonds to the indole ring
These two pockets are separated by a span of ~24
This region has some
elasticity and is
roughly the same distance as the amino acid linker between the
two inserting tryptophan
of many GW proteins. It is thought that argonaute recognizes GW
through identification of tandem tryptophan, separated by this distance
(Schirle & MacRae, 2012).
Filipowicz, W. (2005). RNAi: The
Nuts and Bolts of the RISC Machine. Cell, 122(1), 17–20.
Meister, G., Landthaler, M.,
Patkaniowska, A., Dorsett, Y., Teng, G., & Tuschl, T. (2004).
Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs.
Molecular Cell, 15(2), 185–197.
Schirle, N. T., & MacRae, I.
J. (2012). The
Crystal Structure of Human Argonaute2. Science , 336
(6084 ), 1037–1040. doi:10.1126/science.1221551
Rivas, F. V, Tolia, N. H., Song, J.-J.,
Aragon, J. P., Liu, J., Hannon, G. J., & Joshua-Tor, L. (2005).
Purified Argonaute2 and an siRNA form recombinant human RISC. Nat
Struct Mol Biol, 12(4), 340–349. Retrieved from
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