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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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Processing of 2A-2B is a eukaryote-specific, co-translational event.A, Western blots (WB) probed with anti-HA or anti-FLAG antibody show protein products from HA-2A-2B-FLAG plasmids translated in eukaryotic (HeLa, HeLa cell extract; Retic, rabbit reticulocyte lysate; WG, wheat germ extract) and prokaryotic (S30, bacterial extract; PURE, PURE system) transcription/translation systems. B, Western blots probed with the anti-FLAG antibody show protein products after the PURE system (18 μl) was incubated with the plasmid encoding HA-2A-2B-FLAG in the presence of a HeLa cell extract (1 μl; lanes 1, buffer alone; lanes 2, 10 μg of protein; lanes 3, 20 μg of protein; lanes 4, 40 μg of protein). The HeLa cell extract was added to the system before (left panel) or after (right panel) the translation reaction. In the latter case, incubation was further continued for 2 h at 37 °C. The arrowhead indicates the position of 2B-FLAG.
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Figure 1: Processing of 2A-2B is a eukaryote-specific, co-translational event.A, Western blots (WB) probed with anti-HA or anti-FLAG antibody show protein products from HA-2A-2B-FLAG plasmids translated in eukaryotic (HeLa, HeLa cell extract; Retic, rabbit reticulocyte lysate; WG, wheat germ extract) and prokaryotic (S30, bacterial extract; PURE, PURE system) transcription/translation systems. B, Western blots probed with the anti-FLAG antibody show protein products after the PURE system (18 μl) was incubated with the plasmid encoding HA-2A-2B-FLAG in the presence of a HeLa cell extract (1 μl; lanes 1, buffer alone; lanes 2, 10 μg of protein; lanes 3, 20 μg of protein; lanes 4, 40 μg of protein). The HeLa cell extract was added to the system before (left panel) or after (right panel) the translation reaction. In the latter case, incubation was further continued for 2 h at 37 °C. The arrowhead indicates the position of 2B-FLAG.

Mentions: To examine the mechanism of EMCV 2A-2B processing, various cell-free transcription/translation systems derived from both eukaryotic and prokaryotic cells were programmed with plasmids that encoded the 2A-2B sequence with an N-terminal HA tag and a C-terminal FLAG tag (HA-2A-2B-FLAG). The translated protein products were resolved by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-FLAG antibodies. The HA-2A and 2B-FLAG proteins were separately generated in the eukaryotic systems derived from a HeLa cell extract, a rabbit reticulocyte lysate, and a wheat germ extract (Fig. 1A). In contrast, two prokaryotic cell-free translation systems, S30 (derived from bacterial lysate) and PURE (reconstituted with bacterial translation factors), failed to generate individual 2A and 2B proteins. Instead, they predominantly yielded the unprocessed product (HA-2A-2B-FLAG). Although the S30 system generated several anti-HA-reactive bands that migrated faster than HA-2A-2B-FLAG, this presumably resulted from premature translation termination (Fig. 1A). These results suggested that the 2A-2B processing of EMCV was a eukaryote-specific event, confirming previous results (30).


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)

Processing of 2A-2B is a eukaryote-specific, co-translational event.A, Western blots (WB) probed with anti-HA or anti-FLAG antibody show protein products from HA-2A-2B-FLAG plasmids translated in eukaryotic (HeLa, HeLa cell extract; Retic, rabbit reticulocyte lysate; WG, wheat germ extract) and prokaryotic (S30, bacterial extract; PURE, PURE system) transcription/translation systems. B, Western blots probed with the anti-FLAG antibody show protein products after the PURE system (18 μl) was incubated with the plasmid encoding HA-2A-2B-FLAG in the presence of a HeLa cell extract (1 μl; lanes 1, buffer alone; lanes 2, 10 μg of protein; lanes 3, 20 μg of protein; lanes 4, 40 μg of protein). The HeLa cell extract was added to the system before (left panel) or after (right panel) the translation reaction. In the latter case, incubation was further continued for 2 h at 37 °C. The arrowhead indicates the position of 2B-FLAG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Processing of 2A-2B is a eukaryote-specific, co-translational event.A, Western blots (WB) probed with anti-HA or anti-FLAG antibody show protein products from HA-2A-2B-FLAG plasmids translated in eukaryotic (HeLa, HeLa cell extract; Retic, rabbit reticulocyte lysate; WG, wheat germ extract) and prokaryotic (S30, bacterial extract; PURE, PURE system) transcription/translation systems. B, Western blots probed with the anti-FLAG antibody show protein products after the PURE system (18 μl) was incubated with the plasmid encoding HA-2A-2B-FLAG in the presence of a HeLa cell extract (1 μl; lanes 1, buffer alone; lanes 2, 10 μg of protein; lanes 3, 20 μg of protein; lanes 4, 40 μg of protein). The HeLa cell extract was added to the system before (left panel) or after (right panel) the translation reaction. In the latter case, incubation was further continued for 2 h at 37 °C. The arrowhead indicates the position of 2B-FLAG.
Mentions: To examine the mechanism of EMCV 2A-2B processing, various cell-free transcription/translation systems derived from both eukaryotic and prokaryotic cells were programmed with plasmids that encoded the 2A-2B sequence with an N-terminal HA tag and a C-terminal FLAG tag (HA-2A-2B-FLAG). The translated protein products were resolved by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-FLAG antibodies. The HA-2A and 2B-FLAG proteins were separately generated in the eukaryotic systems derived from a HeLa cell extract, a rabbit reticulocyte lysate, and a wheat germ extract (Fig. 1A). In contrast, two prokaryotic cell-free translation systems, S30 (derived from bacterial lysate) and PURE (reconstituted with bacterial translation factors), failed to generate individual 2A and 2B proteins. Instead, they predominantly yielded the unprocessed product (HA-2A-2B-FLAG). Although the S30 system generated several anti-HA-reactive bands that migrated faster than HA-2A-2B-FLAG, this presumably resulted from premature translation termination (Fig. 1A). These results suggested that the 2A-2B processing of EMCV was a eukaryote-specific event, confirming previous results (30).

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