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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.

Machida K, Mikami S, Masutani M, Mishima K, Kobayashi T, Imataka H - J. Biol. Chem. (2014)

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.

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus

Expression of various proteins with the translation system reconstituted with human factors. The components purified in Fig. 4 were combined with T7 RNA polymerase and programmed with a plasmid encoding Rluc-HA, β-actin-HA, PABP-HA, or β-gal-HA (A) or encoding HA-Rluc, HA-β-actin, HA-PABP, or HA-β-gal (B). Western blotting (WB) was performed with the anti-HA antibody after SDS-PAGE (10%).
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Figure 5: Expression of various proteins with the translation system reconstituted with human factors. The components purified in Fig. 4 were combined with T7 RNA polymerase and programmed with a plasmid encoding Rluc-HA, β-actin-HA, PABP-HA, or β-gal-HA (A) or encoding HA-Rluc, HA-β-actin, HA-PABP, or HA-β-gal (B). Western blotting (WB) was performed with the anti-HA antibody after SDS-PAGE (10%).

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.


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.

Machida K, Mikami S, Masutani M, Mishima K, Kobayashi T, Imataka H - J. Biol. Chem. (2014)

Expression of various proteins with the translation system reconstituted with human factors. The components purified in Fig. 4 were combined with T7 RNA polymerase and programmed with a plasmid encoding Rluc-HA, β-actin-HA, PABP-HA, or β-gal-HA (A) or encoding HA-Rluc, HA-β-actin, HA-PABP, or HA-β-gal (B). Western blotting (WB) was performed with the anti-HA antibody after SDS-PAGE (10%).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231674&req=5

Figure 5: Expression of various proteins with the translation system reconstituted with human factors. The components purified in Fig. 4 were combined with T7 RNA polymerase and programmed with a plasmid encoding Rluc-HA, β-actin-HA, PABP-HA, or β-gal-HA (A) or encoding HA-Rluc, HA-β-actin, HA-PABP, or HA-β-gal (B). Western blotting (WB) was performed with the anti-HA antibody after SDS-PAGE (10%).
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus