Dna End Tag Kit, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation"
Article Title: A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation
Figure Legend Snippet: Schematic of the single-molecule colocalization experiments. ( A ) 80S complexes (with yeast ribosomes) were assembled on IRES (Cy3) molecules (hybridized to a biotinylated DNA) that were tethered to microscope slide surfaces coated with polyethylene glycol (PEG)-Biotin via a streptavidin bridge. In the cartoon shown here, the wild-type IRES has undergone the first pseudotranslocation event so that domain 3 sits at the P site and the Phe-tRNA Phe (Cy5) is delivered to the A site by eEF1A (tRNA from Escherichia coli ), thereby generating a surface-tethered complex with spatially colocalized Cy3 and Cy5 spots. Sample Cy3 and Cy5 frames from an experiment in which Phe-tRNA Phe (Cy5) was delivered as a ternary complex with eEF1A and GTP, to yeast 80S complexes assembled on WT Cricket Paralysis Virus (CrPV) IRES (Cy3), are depicted in ( B ) and ( C ), respectively. The imaged Cy3 and Cy5 spots in these frames are false colored as green and red, respectively. ( D ) Superposition of the two frames in which regions that appear to have colocalized green and red spots, just by manual inspection, are false colored as yellow for visual clarity; the actual analysis of the extent of colocalization involves a much more rigorous mathematical treatment of the raw data using home-built codes. The panels below ( B )–( D ) show a representative region from the corresponding frames, magnified 6x, to demonstrate the well-resolved distribution of spots and the precision of colocalization. ( E ) Identical images as ( B – D ), except in the presence of eEF2, which results in higher levels of colocalization. DOI: http://dx.doi.org/10.7554/eLife.08146.020
Techniques Used: Microscopy