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
Related in: MedlinePlus
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).
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.