Journal: Mol Cell / Year: 2017 Title: Structural and Functional Insights into Human Re-initiation Complexes. Authors: Melanie Weisser / Tanja Schäfer / Marc Leibundgut / Daniel Böhringer / Christopher Herbert Stanley Aylett / Nenad Ban / Abstract: After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large ...After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.
Mass: 18.015 Da / Num. of mol.: 134 / Source method: isolated from a natural source / Formula: H2O
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.73 Å3/Da / Density % sol: 54.89 %
Crystal grow
Temperature: 292 K / Method: vapor diffusion, sitting drop / pH: 7.6 Details: 1:1 mix protein (50 mM HEPES-KOH pH 7.6, 150 mM KCl, 1 mM TCEP) with reservoir solution (100 mM HEPES-KOH pH 7.6, 25 mM sodium formate, 25 mM ammonium acetate, 25 mM sodium citrate tribasic, ...Details: 1:1 mix protein (50 mM HEPES-KOH pH 7.6, 150 mM KCl, 1 mM TCEP) with reservoir solution (100 mM HEPES-KOH pH 7.6, 25 mM sodium formate, 25 mM ammonium acetate, 25 mM sodium citrate tribasic, 25 mM sodium potassium tartrate, 10.7% PEG3350, 10.7% MPD, 10.7% PEG1000)
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