Structure and nucleic-acid binding of the Drosophila Argonaute 2

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Structure and nucleic-acid binding of the Drosophila Argonaute 2
Structure and nucleic-acid
binding of the Drosophila
Argonaute 2 PAZ domain
Andreas Lingel, Bernd Simon, Elisa Izaurralde & Michael
Sattler
European Molecular Biology Laboratory, Meyerhofstrasse 1, D69117 Heidelberg, Germany
Nature. 2003 Nov 27;426(6965): 465-9
PDB ID: 1VYN
指導老師:鄒文雄
主講人:蔡榮育
Introduction

RNA interference (RNAi): This
evolutionary conserved pathway is
triggered in response to exogenous
dsRNA introduced to cells.

For example, by viral infection or
transfection of in vitro synthesized dsRNA,
as well as by the expression of
endogenously encoded RNAi triggers,
microRNAs (miRNA).

RNAi results in the downregulation
of a homologous target gene through
the cleavage, translational
repression, or transcriptional
inhibition of its mRNA.

Two core proteins are universally
associated with RNAi-related
silencing phenomena: Dicer and
Argonaute (Ago).
RNA interference pathways in different organisms
RISC:RNA-induced silencing complex RITS:RNA-induced transcriptional silencing
RNP:ribonucleoprotein
rasiRNA:repeat-associated short interfering RNA
Gunter Meister et. al (2004) Nature Vol.431, 343-349
The siRNA pathway

Members of the Dicer and Argonaute
protein families are essential
components of these RNA-silencing
pathways.

These two families possess an
evolutionarily conserved PAZ
(Piwi/Argonaute/Zwille) domain
whose biochemical function is
unknown.
View structure
Ribbon representation of
the Ago2 PAZ domain
Biological question

The Argonaute 2 protein (Ago2) is a
critical component of RISC. Both
Argonaute and Dicer family
proteins contain a common PAZ
domain whose function is unknown.
(1) What is the function of the PAZ
domain?
(2) Which residue is important?

A region comprising the β3, β4, α3 module
and the central β-barrel, which together
form a clamp-like structure. The presence
of aromatic and positively charged
residues at this surface may indicate a
conserved function in the binding of
negatively charged ligands, such as
nucleic acids.

The PAZ domain contains a variant of the
OB-fold, a module that often binds singlestranded necleic acids ( For example, rho
termination factor in transcription ).
Surface representation of the PAZ
domain coloured by sequence
conservation
Surface representation of the PAZ
domain coloured by electrostatic
charge
White, blue and red corresponds to neutral,
positive and negative electrostatic potential,
respectively.
Nucleic-acid binding activity of the
Ago2 PAZ domain
Result

The RNA-binding region involves highly
conserved residues, specifically Arg 55 (β 2),
Phe 72 (β 3), Thr 80 (β 4), Val 102 (β 5) and
Ile 81 (α 3), but also the less conserved Phe
50 in the β 1–β 2 loop.

The results identify Phe 50 and Phe 72 as
critical residues for RNA binding.

The PAZ domain also binds single-stranded
or double-stranded DNA.
問題探討

PAZ domain 除了 Phe50 和 Phe72 外,還有
哪些 residues 與 RNA 結合?這些 residues
之特性為何?

BLAST 結果呈兩極化,可能之原因為何?

Alignment 結果中其他 conserved residues
可能扮演的角色?

Mapping the electrostatic potential on the
surface of the protein does not yield an
obvious positively charged surface for
nucleic acid binding.

The aromatic residues could interact with
single-stranded nucleic acids via stacking
interactions.

Conserved aromatic residues within the
cleft are involved in RNA binding.
Stereo view showing one PAZ
domain interacting with an siRNA-like
end
Electrostatic surface of PAZ
BLAST
Drosophila melanogaster
 Anopheles gambiae (瘧蚊)
 Homo sapiens
 Gallus gallus
 Caenorhabditis elegans

Alignment
Phe72
Arg31
Asn41 Phe50
Pro91
Pro47
Phe72
Pro66

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