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PDBsum entry 3hax
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Isomerase/biosynthetic protein/RNA
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PDB id
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3hax
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Contents |
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329 a.a.
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53 a.a.
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121 a.a.
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References listed in PDB file
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Key reference
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Title
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Structural mechanism of substrate RNA recruitment in h/aca RNA-Guided pseudouridine synthase.
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Authors
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J.Duan,
L.Li,
J.Lu,
W.Wang,
K.Ye.
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Ref.
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Mol Cell, 2009,
34,
427-439.
[DOI no: ]
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PubMed id
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Abstract
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H/ACA RNAs form ribonucleoprotein complex (RNP) with proteins Cbf5, Nop10, L7Ae,
and Gar1 and guide site-specific conversion of uridine into pseudouridine in
cellular RNAs. The crystal structures of H/ACA RNP with substrate bound at the
active site cleft reveal that the substrate is recruited through
sequence-specific pairing with guide RNA and essential protein contacts.
Substrate binding leads to a reorganization of a preset pseudouridylation pocket
and an adaptive movement of the PUA domain and the lower stem of the H/ACA RNA.
Moreover, a thumb loop flips from the Gar1-bound state in the substrate-free RNP
structure to tightly associate with the substrate. Mutagenesis and enzyme
kinetics analysis suggest a critical role of Gar1 and the thumb in substrate
turnover, particularly in product release. Comparison with tRNA Psi55 synthase
TruB reveals the structural conservation and adaptation between an RNA-guided
and stand-alone pseudouridine synthase and provides insight into the
guide-independent activity of Cbf5.
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Figure 1.
Figure 1. Overview of Substrate-Bound H/ACA RNP Structures
(A) Secondary structure of H/ACA RNA1 with substrate RNA
bound at the pseudouridylation pocket. The lower (P1) and upper
(P2) stems, substrate-guide helices PS1 and PS2, and the lower
(J1) and upper (J2) three-way junctions are indicated. Base pair
symbols are depicted according to a previous rule (Leontis and
Westhof, 2001). Hollow letters represent disordered residues in
the Gar-minus structure. Prime denotes substrate RNA.
(B)
Ribbon representation of the substrate-bound Gar1-minus H/ACA
RNP structure, showing Cbf5 PUA domain in light green; Cbf5
catalytic (Cat) domain in dark green; Nop10 in magenta; L7Ae in
blue; substrate RNA in purple; guide sequences in orange; the
ACA motif, kink-turn motif, and target nucleotide in red; and
the remainder of the H/ACA RNA in yellow. The target nucleotide
is shown as sticks.
(C) SIGMAA-weighted 2F[o] − F[c]
electron density map of the Gar1-minus complex. The 2.1 Å
map is contoured at 1 σ. The refined structure is shown as
sticks with carbon atoms colored in pink, oxygen in red,
nitrogen in blue, phosphorus in orange, and magnesium in green.
(D) The substrate-bound full-complex structure represented
as Cα or P traces. The solvent-modified 5 Å electron
density map contoured at 1 σ is indicated. Gar1 is colored
cyan, and other parts are color coded as in Figure 1B. A
symmetry-related molecule, colored in gray, forms a self-duplex
with the 5′ extension of H/ACA RNA2.
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Figure 2.
Figure 2. Structure and RNP Assembly of Substrate-Bound H/ACA
RNA
(A) Structure of substrate-bound H/ACA RNA. Hydrated Mg
ions are shown as sticks with Mg colored green and water red.
(B) RNA-binding surface in substrate-bound H/ACA RNP. Shown
are the Gar1-minus RNP structure with Gar1 modeled according to
its position in the full complex. RNAs are represented as
ribbons and planes, and proteins are represented as surfaces.
Individual parts are color coded as in Figure 1B. The
RNA-binding surface (within 4 Å of RNA) is colored in gray.
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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2009,
34,
427-439)
copyright 2009.
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