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PDBsum entry 1q2s
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Transferase/RNA
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PDB id
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1q2s
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate.
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Authors
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W.Xie,
X.Liu,
R.H.Huang.
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Ref.
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Nat Struct Biol, 2003,
10,
781-788.
[DOI no: ]
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PubMed id
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Abstract
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Prokaryotic tRNA guanine transglycosylase (TGT) catalyzes replacement of guanine
(G) by 7-aminomethyl-7-deazaguanine (PreQ1) at the wobble position of four
specific tRNAs. Addition of 9-deazaguanine (9dzG) to a reaction mixture of
Zymomonas mobilis TGT and an RNA substrate allowed us to trap, purify and
crystallize a chemically competent covalent intermediate of the TGT-catalyzed
reaction. The crystal structure of the TGT-RNA-9dzG ternary complex at a
resolution of 2.9 A reveals, unexpectedly, that RNA is tethered to TGT through
the side chain of Asp280. Thus, Asp280, instead of the previously proposed
Asp102, acts as the nucleophile for the reaction. The RNA substrate adopts an
unusual conformation, with four out of seven nucleotides in the loop region
flipped out. Interactions between TGT and RNA revealed by the structure provide
the molecular basis of the RNA substrate requirements by TGT. Furthermore,
reaction of PreQ1 with the crystallized covalent intermediate provides insight
into the necessary structural changes required for the TGT-catalyzed reaction to
occur.
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Figure 3.
Figure 3. Overall structure of the trapped covalent
intermediate. (a) Ribbon representation of the TGT -RNA -9dzG
ternary complex. TGT is colored according to the assignment of
the secondary structures in Figure 2. The RNA is dark green.
9dzG is shown in ball-and-stick model in red, and a zinc ion is
represented by a ball in magenta. (b) Summary of TGT -RNA, TGT
-9dzG and RNA -RNA interactions. TGT, RNA and 9dzG are colored
as in a except the phosphate groups of RNA are in magenta. The
conserved RNA nucleotides 33 -35, 9dzG and Asp280 are
highlighted with color-filled boxes. Arrows in blue represent
hydrogen bonds between RNA and TGT, RNA and water, or 9dzG and
TGT. Arrows in red show stacking of amino acid side chains from
TGT on bases of RNA or on 9dzG. Two arrows in green represent
intramolecular interactions in RNA. 'W' in a circle represents
well-positioned water molecules in the structure.
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Figure 5.
Figure 5. The active site and molecular basis of substrate
specificity. (a) Stereo view of TGT active site. The main
chains of TGT are grayish blue, the side chains of TGT are
orange and the RNA and 9dzG are green. The hetero-atoms are
colored individually, with nitrogen in blue, oxygen in red and
phosphate in magenta. The C9 of 9dzG is highlighted in black. A
water molecule is shown as a small ball in red and labeled 'W'.
Hydrogen bonds are indicated with dashed lines. The side chains
of Asp106 and Cys158, which block 9dzG from the top, are omitted
for clarity. (b,c) Recognition of U33 (b) and U35 (c) in RNA
substrate by TGT. The color schemes are the same as in a. The
structure of the phosphate group of A38 in c is omitted but
labeled for clarity.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Biol
(2003,
10,
781-788)
copyright 2003.
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