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: The 2A fragment might be generated due to a translational pause of the ribosome at the C terminus of the 2A region. In that case, the 2A protein should remain ligated to the tRNAGly in the ribosomal P site as demonstrated in SecM-mediated ribosomal stall (31), translational pause in the XBP1u mRNA (32), and ribosomal arrest mediated by the upstream open reading frame 2 (uORF2) of the cytomegalovirus UL 4 gene (33). To examine this, we incubated the HeLa cell extract with the HA-2A-2B-FLAG construct and resolved the product with NuPAGE (Invitrogen; the pH of the gel is neutral) (34) to maximally retain the esterified bond between the 3′-end of the tRNA and the C terminus of the 2A peptide. Western blotting with anti-HA antibody detected the 2A peptide (Fig. 3, lane 1). We tested a positive control with a plasmid encoding HA-2A(uORF2) in which the C-terminal 22 amino acids of 2A had been replaced with uORF2 of the cytomegalovirus UL 4 gene (33). From this template, a product that migrated slower than the HA-2A peptide was generated (lane 2). Treatment with RNase A eliminated this slowly migrating product (lane 4). This product was also abolished by treatment with peptidyl-tRNA hydrolase (Ref. 35; kindly provided by Dr. Nagao) (we confirmed that the peptidyl-tRNA hydrolase preparation used in this experiment was devoid of an RNase activity that would decompose the body of tRNA; data not shown), indicating that the peptide was conjugated to tRNA. From the HA-2A-2B-FLAG construct, neither RNase A- nor peptidyl-tRNA hydrolase-sensitive product was produced (Fig. 3, lanes 1, 3, and 5). Thus, the 2A peptide was dissociated from tRNA despite the absence of a termination codon.
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.