37 research outputs found

    Isothermal calorimetric titration of eIF3j with eIF3b-RRM.

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    <p>The <i>upper panel</i> shows raw data of heat effect (in µcal·s<sup>−1</sup>) of 20-µl injections of 229 µM eIF3b-RRM into 1.5 ml of 10 µm eIF3j performed at 300 s intervals. The <i>lower panel</i> shows the fitted binding isotherms. The data points were obtained by integration of heat signals plotted against the molar ratio of eIF3b-RRM to eIF3j in the reaction cell. The <i>solid line</i> represents a calculated curve using the best fit parameters obtained by a nonlinear least squares fit.</p

    Sequence-based alignment of human and yeast eIF3j.

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    <p>The boxed sequence is the fragment which was solved in complex with human eIF3b-RRM (PDB code 2KRB). Conservation of the sequence of this region suggests the same mode of binding between yeast eIF3j and eIF3b-RRM. The alignment and the color representation were performed using ClustalW2 (<a href="http://www.ebi.ac.uk/Tools/clustalw2" target="_blank">www.ebi.ac.uk/Tools/clustalw2</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012784#pone.0012784-Larkin1" target="_blank">[24]</a> and ESPript 2.2 (<a href="http://www.espript.ibcp.fr/ESPript" target="_blank">www.espript.ibcp.fr/ESPript</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012784#pone.0012784-Gouet1" target="_blank">[25]</a> respectively.</p

    Summary of the data collection and structure refinement.

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    <p>Values in brackets refer to the highest resolution shell.</p>a<p>R<sub>merge</sub>  =  Σ<sub>h</sub>Σ<sub>l</sub> | I<sub>ih</sub>-h> |/Σ<sub>h</sub>Σ<sub>I</sub> h>, where h> is the mean of the observations I<sub>ih</sub> of reflection h.</p>b<p>AU stands for asymmetric unit.</p>c<p>R<sub>work</sub>  =  Σ<sub>hkl</sub>| |F<sub>obs</sub>|–|F<sub>calc</sub>| |/Σ<sub>hkl</sub> |F<sub>obs</sub>|, where F<sub>obs</sub> and F<sub>calc</sub> are the observed and calculated structure factors, respectively.</p>d<p>R factor calculated for 5% randomly chosen reflections not included in the refinement.</p>e<p>The geometry of the models was analyzed by Molprobity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012784#pone.0012784-Davis1" target="_blank">[26]</a>.</p

    Critical interactions between two monomers in the asymmetric unit mimic the presence of an oligonucleotide.

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    <p>(A). The dimer of the RRM<sup>76–170</sup> is held by several interactions, some of which involve components of the RNP1 (Lys 124, Phe 126 and Phe 128) or RNP2 (Asn 82). (B) Superposition of eIF3b-RRM with hnRNP A1 (UP1) in complex with single-stranded telomeric DNA (PDB code 2UP1) shows that different elements of RNP1 and RNP2 can interact with RNA bases (Asn82 in one of its alternative conformations and Phe128), sugar pocket (Phe126) or phosphate backbone (Lys124). (C) The interacting partners for the residues depicted in panel B assume the position of certain elements of the docked oligonucleotide; e.g. Asp 162 sits where the negatively charged phosphate group would sit, Phe 156 and Phe 166 occupy the position of bases of the oligonucleotide and Tyr 158 mimics the sugar pocket.</p

    Binding studies between yeast IF3b-RRM and total RNA extract.

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    <p>(A). Electrophoretic mobility shift assay between RRM and total RNA indicates the upwards smearing of the RRM band upon interaction with RNA. The effect is more pronounced at lower salt concentration. (B). Filter binding assay confirms the results obtained by EMSA. Drops of filter binding assay are aligned under the corresponding lanes of EMSA. * stands for mM.</p

    Surface charge distribution of yeast and human eIF3b-RRM.

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    <p>(A) Surface charge distribution of yeast eIF3b-RRM viewed from the α-helical side of the domain (distal to the oligonucleotide binding side) showing the accumulation of positive charges which would provide a suitable binding site for the negatively charged peptide of eIF3j. (B) Surface charge distribution of human eIF3b-RRM from the same view as (A). The short negatively charged peptide of eIF3j (magenta) sits in a basic cleft on eIF3b-RRM. (C). Surface charge distribution on the nucleotide binding side of the yeast eIF3b-RRM indicates the dominance of the positive over negative charges. This suggests that this motif can accommodate oligonucleotides. (D). Surface charge distribution on the nucleotide binding side of the humaneIF3b-RRM. Accumulation of acidic side-chains leaves no room for accommodation of any oligonucleotide.</p

    Different dimer formation between two different truncations of yeast eIF3b-RRM.

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    <p>Superimposition of the chains A of the yeast RRM<sup>76–161</sup> (red) and RRM<sup>76–170</sup> (green) indicates the difference in the relative orientation of the dimers between two molecules, indicating that the formation of the dimer is probably an artifact of the crystallization.</p

    Overall structure of yeast eIF3b-RRM.

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    <p>(A) Overall fold of the yeast eIF3b-RRM showing the canonical β-α-β-β-α-β fold which at the C-terminus is followed by an extra helix which connects this domain to the rest of the protein (N- and C-termini are colored blue and red, respectively). (B) Relative orientation of two monomers in the asymmetric unit. Two monomers are held in place by interactions between residues in their β-sheets as well as the c-terminal helices. A 180° non-crystallographic symmetry axis exists between two monomers.</p

    Superimposition of yeast eIF3b-RRM with several canonical RRMs bound to oligonucleotides from PDB.

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    <p>(A) Six different canonical RRMs (PDB codes 1B7F, 2AD9, 2KH9, 2RQC, 3D2W and 2UP1, all in grey) superimposed with yeast eIF3b-RRM (orange). As shown, oligonucleotides (red loops) occupy more or less the same position on the solvent exposed side of the β-sheet. (B) Three dimensional conservation of the elements of RNP1 (the black box on the panel A). The numbers correspond to the position of the amino acids in the motif [RK]<sub>1</sub>-G<sub>2</sub>-[FY]<sub>3</sub>-[GA]<sub>4</sub>-[FY]<sub>5</sub>-[ILV]<sub>6</sub>-X<sub>7</sub>-[FY]<sub>8</sub>.</p

    Structural comparison of <i><sup>Dm</sup></i>cN-IIIB and <i><sup>Mm</sup></i>cN-IIIA.

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    <p>Detail view on the substrate-binding pockets of <i><sup>Dm</sup></i>cN-IIIB bound to m<sup>7</sup>guanosine in red (A) and <i><sup>Mm</sup></i>cN-IIIA bound to UMP in dark blue (B). The left panel shows the binding pocket with labeled residues and substrates represented as sticks. Note that Phe75 of <i><sup>Dm</sup></i>cN-IIIB is replaced by a histidine in <i><sup>Hs</sup></i>cN-IIIA while the Trp121 of <i><sup>Dm</sup></i>cN-IIIB is replaced by a tyrosine in <i><sup>Hs</sup></i>cN-IIIA. In the right panel the substrate-binding pockets are shown as surfaces, which were calculated using a probe radius of 1.65 Å and for clarity reasons were clipped at the level of the nucleobases. Note that the substrate-binding pocket for m<sup>7</sup>G (cN-IIIB, red surface) is significantly larger than the UMP binding pocket of cN-IIIA (blue surface), which was thought to be the structural reason for its purine substrate exclusion. (C) Orientation of substrate binding residues and the corresponding pocket after docking m<sup>7</sup>GMP to <i><sup>Mm</sup></i>cN-IIIA (light blue). Note that Asn69 (marked by an asterisk) rotates by 180 degrees in comparison to (B) and adopts another common rotamer thus enlarging the binding pocket and neatly accommodating the m<sup>7</sup>GMP.</p
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