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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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2A-2B processing does not require eRFs.A, the plasmid pUC-T7-HCV-HA-2A-2B-HA was incubated in a HeLa cell extract-derived protein synthesis system (left panel) or in our reconstituted protein expression system with eRFs (right panel). B, the HeLa cell extract-derived protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA. C, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs. D, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs as in C, but the expression system was scaled up 2-fold. E, the reconstituted protein expression system was incubated with a template encoding HA-2A-HA-2B (22 aa) in the presence (+) or absence (−) of eRFs. In all cases, Western blotting (WB) was performed with anti-HA antibody after SDS-PAGE (12.5%).
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Figure 7: 2A-2B processing does not require eRFs.A, the plasmid pUC-T7-HCV-HA-2A-2B-HA was incubated in a HeLa cell extract-derived protein synthesis system (left panel) or in our reconstituted protein expression system with eRFs (right panel). B, the HeLa cell extract-derived protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA. C, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs. D, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs as in C, but the expression system was scaled up 2-fold. E, the reconstituted protein expression system was incubated with a template encoding HA-2A-HA-2B (22 aa) in the presence (+) or absence (−) of eRFs. In all cases, Western blotting (WB) was performed with anti-HA antibody after SDS-PAGE (12.5%).

Mentions: After establishing the protein synthesis system reconstituted with human factors, we programmed this system with a template that encoded HA-2A-2B-HA. Western blots probed with anti-HA antibody showed that, like the HeLa cell extract, the reconstituted system was able to process the 2A-2B peptides (Fig. 7A); the upper and lower bands correspond to 2A-HA and 2B-HA peptides, respectively (Fig. 7B). To examine whether eRFs were required for the processing, eRFs were removed from the reconstituted system and programmed with plasmids encoding HA-2A-2B-HA, HA-2A-2B-FLAG, and FLAG-2A-2B-HA. Western blots were probed with anti-HA antibody (Fig. 7C). Synthesis of 2A remained unchanged upon withdrawal of eRFs, but the 2B-HA band was up-shifted, and the intensity of the 2B-HA band of the original mobility was decreased. The 2B-HA-related products were hardly seen on the blot of Fig. 7C, but they were clearly detectable in the experiments that were scaled up (Fig. 7D). These results suggested that translation of 2A terminated, even when eRFs were omitted, despite the absence of a termination codon; however, translation of 2B-HA terminated at the authentic termination codon, and therefore its translation termination required eRFs. Hence, we constructed a template, HA-2A-HA-2B (22 aa), in which the C-terminal 22 amino acids of 2B were replaced with the C-terminal 20 amino acids of 2A (AHYAG----TNPG) and the N-terminal two amino acids of 2B (PF); this construct was designed to allow the translation of 2B to terminate in the same manner as 2A. Then, to detect the 2B product, we added an HA tag to the N terminus of 2B. When programmed with this template, 2B and 2A were both generated in the presence or absence of eRFs (Fig. 7E). These results indicate that 2A-2B processing requires neither eIFs nor eRFs and occurs during the elongation process carried out by the ribosomes, eEF1s, and eEF2.


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)

2A-2B processing does not require eRFs.A, the plasmid pUC-T7-HCV-HA-2A-2B-HA was incubated in a HeLa cell extract-derived protein synthesis system (left panel) or in our reconstituted protein expression system with eRFs (right panel). B, the HeLa cell extract-derived protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA. C, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs. D, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs as in C, but the expression system was scaled up 2-fold. E, the reconstituted protein expression system was incubated with a template encoding HA-2A-HA-2B (22 aa) in the presence (+) or absence (−) of eRFs. In all cases, Western blotting (WB) was performed with anti-HA antibody after SDS-PAGE (12.5%).
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Figure 7: 2A-2B processing does not require eRFs.A, the plasmid pUC-T7-HCV-HA-2A-2B-HA was incubated in a HeLa cell extract-derived protein synthesis system (left panel) or in our reconstituted protein expression system with eRFs (right panel). B, the HeLa cell extract-derived protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA. C, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA, HA-2A-2B-FLAG, or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs. D, the reconstituted protein expression system was incubated with a template encoding HA-2A-2B-HA or FLAG-2A-2B-HA in the presence (+) or absence (−) of eRFs as in C, but the expression system was scaled up 2-fold. E, the reconstituted protein expression system was incubated with a template encoding HA-2A-HA-2B (22 aa) in the presence (+) or absence (−) of eRFs. In all cases, Western blotting (WB) was performed with anti-HA antibody after SDS-PAGE (12.5%).
Mentions: After establishing the protein synthesis system reconstituted with human factors, we programmed this system with a template that encoded HA-2A-2B-HA. Western blots probed with anti-HA antibody showed that, like the HeLa cell extract, the reconstituted system was able to process the 2A-2B peptides (Fig. 7A); the upper and lower bands correspond to 2A-HA and 2B-HA peptides, respectively (Fig. 7B). To examine whether eRFs were required for the processing, eRFs were removed from the reconstituted system and programmed with plasmids encoding HA-2A-2B-HA, HA-2A-2B-FLAG, and FLAG-2A-2B-HA. Western blots were probed with anti-HA antibody (Fig. 7C). Synthesis of 2A remained unchanged upon withdrawal of eRFs, but the 2B-HA band was up-shifted, and the intensity of the 2B-HA band of the original mobility was decreased. The 2B-HA-related products were hardly seen on the blot of Fig. 7C, but they were clearly detectable in the experiments that were scaled up (Fig. 7D). These results suggested that translation of 2A terminated, even when eRFs were omitted, despite the absence of a termination codon; however, translation of 2B-HA terminated at the authentic termination codon, and therefore its translation termination required eRFs. Hence, we constructed a template, HA-2A-HA-2B (22 aa), in which the C-terminal 22 amino acids of 2B were replaced with the C-terminal 20 amino acids of 2A (AHYAG----TNPG) and the N-terminal two amino acids of 2B (PF); this construct was designed to allow the translation of 2B to terminate in the same manner as 2A. Then, to detect the 2B product, we added an HA tag to the N terminus of 2B. When programmed with this template, 2B and 2A were both generated in the presence or absence of eRFs (Fig. 7E). These results indicate that 2A-2B processing requires neither eIFs nor eRFs and occurs during the elongation process carried out by the ribosomes, eEF1s, and eEF2.

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