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: To delineate the minimal region of 2A-2B needed for processing, we created serial truncations that removed different lengths of the N-terminal region of 2A in the HA-2A-2B-FLAG construct. Translation in the HeLa cell extract showed that the C-terminal 18 amino acids of 2A were the minimum required for processing (Fig. 2A). Next, we created alanine substitutions for each of these 18 amino acids (except two native alanine residues) and for the first four amino acids in 2B. The results verified the importance of the NPGP sequence (the glycine residue is the last amino acid of 2A) for processing (Fig. 2B) (15, 18). We then tested silent mutations for NPGP in which the nucleotide sequence was changed but the NPGP amino acids were maintained. These silent mutations showed full processing efficiency (Fig. 2C). Thus, it was highly unlikely that the mRNA sequence for NPGP was involved in processing.
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.