A translation system reconstituted with human factors proves that processing of encephalomyocarditis virus proteins 2A and 2B occurs in the elongation phase of translation without eukaryotic release factors.
Bottom Line: These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event.We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs.Our results indicate that this unusual event occurs in the elongation phase of translation.
Affiliation: From the Department of Materials Science and Chemistry and Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan and.Show MeSH
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Mentions: To examine the efficiency of this reconstituted system, the system was programmed with a plasmid with an ORF for Rluc, β-actin, PABP, or β-gal placed between the HCV IRES and T7 RNA polymerase terminator sequences in the pUC-T7-HCV IRES vector (20). To detect the full-length product, each protein sequence was tagged with HA at the C terminus. After incubation at 32 °C for 3 h, samples were resolved by SDS-PAGE followed by Western blotting with anti HA antibody. This reconstitution system was able to produce the full-length forms (37–120 kDa) of each encoded protein but tended to produce lower amounts of longer polypeptides (Fig. 5A). When the HA tag was moved to the N terminus of the same ORFs, several shorter products were detected (Fig. 5B). These products were presumably due to premature termination of translation, thereby reducing the yield of the full-length products. Note that because of a high sensitivity of the anti-HA antibody for probing Western blots, only HA-dependent Western blotting was used to monitor protein synthesis with the reconstitution system.
Affiliation: From the Department of Materials Science and Chemistry and Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan and.