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PDBsum entry 3kit
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120 a.a.
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271 a.a.
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204 a.a.
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207 a.a.
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181 a.a.
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164 a.a.
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145 a.a.
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130 a.a.
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138 a.a.
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122 a.a.
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146 a.a.
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140 a.a.
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117 a.a.
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98 a.a.
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135 a.a.
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117 a.a.
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101 a.a.
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113 a.a.
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92 a.a.
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100 a.a.
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184 a.a.
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84 a.a.
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93 a.a.
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71 a.a.
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59 a.a.
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57 a.a.
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55 a.a.
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50 a.a.
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47 a.a.
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63 a.a.
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37 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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The structural basis for mRNA recognition and cleavage by the ribosome-Dependent endonuclease rele.
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Authors
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C.Neubauer,
Y.G.Gao,
K.R.Andersen,
C.M.Dunham,
A.C.Kelley,
J.Hentschel,
K.Gerdes,
V.Ramakrishnan,
D.E.Brodersen.
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Ref.
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Cell, 2009,
139,
1084-1095.
[DOI no: ]
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PubMed id
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Abstract
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Translational control is widely used to adjust gene expression levels. During
the stringent response in bacteria, mRNA is degraded on the ribosome by the
ribosome-dependent endonuclease, RelE. The molecular basis for recognition of
the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here,
we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to
programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A)
cleavage. RelE occupies the A site and causes cleavage of mRNA after the second
nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced
hydrolysis. Stacking of A site codon bases with conserved residues in RelE and
16S rRNA explains the requirement for the ribosome in catalysis and the subtle
sequence specificity of the reaction. These structures provide detailed insight
into the translational regulation on the bacterial ribosome by mRNA cleavage.
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Figure 1.
Figure 1. Overview of the RelE-Bound 70S Ribosome
(A)
Top view of the 70S ribosome with the 50S (blue) and 30S (wheat)
subunits surrounding RelE (A site, blue), tRNA^fMet (P site,
green), a noncognate tRNA^fMet (E site, red), and mRNA
(magenta). (A)–(C) are based on the precleavage structure.
(B) Close-up of the A and P sites of the 30S subunit viewed
from the interface to the 50S. RelE (blue cartoon) spans the 16S
rRNA from the head (helix 31 region, green) to the body (helix
18, pink). The mRNA is shown in purple sticks, and the P and E
site tRNAs colored as in (A).
(C) Close-up view of the A
and P sites showing RelE (blue Cα trace), mRNA (purple sticks),
and P site tRNA (green cartoon) along with the DF[o]-mF[c]
electron density of the precleavage structure contoured at 1.5
σ. The mRNA sequence is indicated.
(D) The postcleavage
structure showing the position of the 2′-3′ cyclic phosphate
generated upon cleavage (2′-3′ cP). The map is contoured at
1.2 σ.
See also Figure S1.
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Figure 3.
Figure 3. In Vitro mRNA Cleavage Assay on the 70S Ribosome
(A) Sequence of E. coli RelE with the conservation among
homologs indicated as increasing strength of red color and the
conserved tyrosine at the C terminus in light blue. The
secondary structure is shown above the sequence and the
interactions to rRNA and mRNA below (all numbers correspond to
the E. coli 16S sequence). Residues in the P. horikoshii RelE
homolog that affect the activity are indicated with black boxes
(Takagi et al., 2005).
(B) Overview of the mRNAs used for
the in vitro cleavage assays. The 25 nt mRNA consists of a
Shine-Dalgarno element (SD) followed by a spacer and the P site
(AUG) and A site (UAG) codons. “Trunc Asite” ends after the
P site codon with a 3′-OH. The table shows predicted masses of
full length mRNA and fragments that would result from cleavage
after position 1 or 2 of the A site codon leaving either a
3′-OH, 3′-phosphate (3′-P), or 2′-3′ cyclic phosphate
(2′,3′-cP).
(C) MALDI mass spectrometry spectra and
masses of RNA fragments isolated from complexes in the absence
(blue) or presence (red) of RelE.
(D) In vitro cleavage
assay using 5′ ^32P-labeled mRNA substrates. • is the 25 nt
unmodified mRNA; MAP has phosphorothioate linkages after A site
codon positions 1 and 2; MAO, MAO2, and MAO3 are
2′-O-methylated at position 1, positions 1 + 2, or all three
positions, respectively; and MAD contains a deoxyribose at
position 1. The mRNAs were incubated with either T. thermophilus
(T.th.) or E. coli (E.co.) 70S ribosomes, tRNA^fMet, and either
RelE^wt or RelE^R81A/R45A (RelE^dm) as indicated for either 1 hr
(lanes 1–16) or overnight (lanes 17 and 18). The size markers
indicate the positions of the full-length (25 nt 3′-OH) and
Trunc Asite (18 nt 3′-OH) RNAs and the 20 nt 2′-3′ cyclic
phosphate cleavage product, which runs approximately 1 nt faster
than the corresponding 3′-OH species.
See also Figure S3.
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The above figures are
reprinted
by permission from Cell Press:
Cell
(2009,
139,
1084-1095)
copyright 2009.
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