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Structural and functional insights into human re-initiation complexes

Molecular Cell. 2017;

Melanie Weisser, Tanja Schafer, € Marc Leibundgut, Daniel Bo¨ hringer, Christopher Herbert Stanley Aylett, Nenad Ban

Products/Services Used	Details	Operation
PCR Cloning and Subcloning	HRP-conjugated anti-His-tag-antibody from mouse (GenScript) was added at a ratio of 1:5000 to a final concentration of 0.1 mg/ml in washing buffer (1x PBS, 0.05% Tween 20, supplemented with 1% (w/v) BSA), followed by shaking for 1h at room temperature.Open reading frames of C-terminally TEV-cleavable 6x-His-tagged human eIF2D (isoform 1), MCT-1 and DENR were commercially synthesized by GenScript USA into pUC57 vectors, amplified via PCR and inserted into pLIC and pLIC-His6 vectors by ligation-independent cloning (adopted from Doyle, 2005).The human eIF2D SWIB/MDM2-SUI construct (comprising residues 380 to 584 of eIF2D) was amplified from the Genscript eIF2DpUC57 vector and inserted into a pLIC-His6 vector by ligation-independent cloning (adopted from Doyle, 2005).HRP-conjugated anti-His-tag-antibody from mouse (GenScript) was added at a ratio of 1:5000 to a final concentration of 0.1 mg/ml in washing buffer (1x PBS, 0.05% Tween 20, supplemented with 1% (w/v) BSA), followed by shaking for 1h at room temperature.	Get A Quote

摘要

Weisser et al. solve the structures of reinitiation factors in complex with the small ribosomal subunit, mRNA, and initiator tRNA. The structures reveal how reinitiation factors monitor start codon recognition and position the initiator tRNA to mimic the functions of canonical translation initiation factors.