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PDBsum entry 2vqf
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235 a.a.
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207 a.a.
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208 a.a.
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151 a.a.
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101 a.a.
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155 a.a.
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138 a.a.
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127 a.a.
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99 a.a.
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119 a.a.
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125 a.a.
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125 a.a.
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60 a.a.
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88 a.a.
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84 a.a.
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104 a.a.
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73 a.a.
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81 a.a.
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99 a.a.
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25 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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Modified uridines with c5-Methylene substituents at the first position of the tRNA anticodon stabilize u.G wobble pairing during decoding.
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Authors
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S.Kurata,
A.Weixlbaumer,
T.Ohtsuki,
T.Shimazaki,
T.Wada,
Y.Kirino,
K.Takai,
K.Watanabe,
V.Ramakrishnan,
T.Suzuki.
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Ref.
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J Biol Chem, 2008,
283,
18801-18811.
[DOI no: ]
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PubMed id
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Abstract
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Post-transcriptional modifications at the first (wobble) position of the tRNA
anticodon participate in precise decoding of the genetic code. To decode codons
that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine
derivatives (xm(5)U) at the wobble position. However, the functional properties
of the C5-substituents of xm(5)U in codon recognition remain elusive. We
previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point
mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic
acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine
(taum(5)U) modification and caused a decoding defect. Here, we constructed
Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the
wobble position and measured their decoding activities in an in vitro
translation as well as by A-site tRNA binding. In addition, the decoding
properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of
the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter
constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U
modification plays a critical role in decoding NNG codons by stabilizing U.G
pairing at the wobble position. Crystal structures of an anticodon stem-loop
containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site
revealed that the taum(5)U.G base pair does not have classical U.G wobble
geometry. These structures provide help to explain how the taum(5)U modification
enables efficient decoding of UUG codons.
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Figure 1.
FIGURE 1. Construction of E. coli tRNA^Leu(UUR) bearing
wobble modifications. A, chemical structures of the modified
uridines introduced into the wobble position of E. coli
tRNA^Leu(UUR): cmnm^5U and  m^5U. B, cloverleaf
structure of E. coli tRNA^Leu(UUR). U^*, the modified uridine.
C, schematic depiction of the molecular surgery procedure used
to construct E. coli tRNA^Leu(UUR) bearing the wobble
modifications. The details are described under "Experimental
Procedures." BAP, bacterial alkaline phosphatase; PNK,
polynucleotide kinase.
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Figure 5.
FIGURE 5. Crystal structures of an  interacting
with either UUA or UUG in the ribosomal A-site. A, unbiased
difference Fourier electron density maps for ASL and mRNA (green
mesh) are shown in stereo for the complex with the UUA codon.
The 5' part of the ASL up to and including parts of U33 is not
visible. B, the same as A but for the complex having a UUG codon
in the A-site. C, the m^5U-A base pair along
with unbiased difference Fourier electron density maps (green
mesh). The base pair refined into a position with slightly
distorted WC geometry. Weak density for the sulfonic acid group
was visible at this  level. D, the m^5U·G base pair
with unbiased difference Fourier electron density maps (green
mesh). Although the density is not very strong, it can be
excluded that the base pair adopts G·U wobble geometry
(compare modeled G·U base pair in transparent gray with
electron density maps). E, comparison of stacking interaction
between a modeled G·U wobble base pair and the m^5U·G base pair.
The modified uridine has a more favorable stacking interaction
with A35 than an unmodified base would have.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
18801-18811)
copyright 2008.
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