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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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Amino acids important for 2A-2B processing.A, the HeLa cell extract-derived protein expression system was programmed with a template encoding an N-terminally truncated 2A-2B-FLAG, pUC-T7-HCV 2A (14, 15, 16, 17, 18, 19, or 20 aa)-2B-FLAG. The number above each panel represents the number of amino acids of 2A after truncation. Western blotting (WB) was performed with anti-FLAG antibody. B, templates bearing an alanine substitution for each of the C-terminal 18 amino acids of 2A (except two native alanine residues) and for the N-terminal four amino acids of 2B in the plasmid pUC-T7-HCV 2A (18 aa)-2B-FLAG were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-FLAG antibody. Each amino acid is numbered from the C-terminal amino acid (glycine) of 2A, which is set to +1. The efficiency of processing (2B-FLAG/2B-FLAG + 2A (18 aa)-2B-FLAG) is reported below each panel. C, plasmids pUC-T7-HCV HA-2A-2B-HA (lane 1), pUC-T7-HCV HA-2A(silent-1)2B-HA (lane 2), and pUC-T7-HCV HA-2A(silent-2)2B-HA (lane 3) were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-HA antibody.
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Figure 2: Amino acids important for 2A-2B processing.A, the HeLa cell extract-derived protein expression system was programmed with a template encoding an N-terminally truncated 2A-2B-FLAG, pUC-T7-HCV 2A (14, 15, 16, 17, 18, 19, or 20 aa)-2B-FLAG. The number above each panel represents the number of amino acids of 2A after truncation. Western blotting (WB) was performed with anti-FLAG antibody. B, templates bearing an alanine substitution for each of the C-terminal 18 amino acids of 2A (except two native alanine residues) and for the N-terminal four amino acids of 2B in the plasmid pUC-T7-HCV 2A (18 aa)-2B-FLAG were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-FLAG antibody. Each amino acid is numbered from the C-terminal amino acid (glycine) of 2A, which is set to +1. The efficiency of processing (2B-FLAG/2B-FLAG + 2A (18 aa)-2B-FLAG) is reported below each panel. C, plasmids pUC-T7-HCV HA-2A-2B-HA (lane 1), pUC-T7-HCV HA-2A(silent-1)2B-HA (lane 2), and pUC-T7-HCV HA-2A(silent-2)2B-HA (lane 3) were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-HA antibody.

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.


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)

Amino acids important for 2A-2B processing.A, the HeLa cell extract-derived protein expression system was programmed with a template encoding an N-terminally truncated 2A-2B-FLAG, pUC-T7-HCV 2A (14, 15, 16, 17, 18, 19, or 20 aa)-2B-FLAG. The number above each panel represents the number of amino acids of 2A after truncation. Western blotting (WB) was performed with anti-FLAG antibody. B, templates bearing an alanine substitution for each of the C-terminal 18 amino acids of 2A (except two native alanine residues) and for the N-terminal four amino acids of 2B in the plasmid pUC-T7-HCV 2A (18 aa)-2B-FLAG were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-FLAG antibody. Each amino acid is numbered from the C-terminal amino acid (glycine) of 2A, which is set to +1. The efficiency of processing (2B-FLAG/2B-FLAG + 2A (18 aa)-2B-FLAG) is reported below each panel. C, plasmids pUC-T7-HCV HA-2A-2B-HA (lane 1), pUC-T7-HCV HA-2A(silent-1)2B-HA (lane 2), and pUC-T7-HCV HA-2A(silent-2)2B-HA (lane 3) were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-HA antibody.
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Figure 2: Amino acids important for 2A-2B processing.A, the HeLa cell extract-derived protein expression system was programmed with a template encoding an N-terminally truncated 2A-2B-FLAG, pUC-T7-HCV 2A (14, 15, 16, 17, 18, 19, or 20 aa)-2B-FLAG. The number above each panel represents the number of amino acids of 2A after truncation. Western blotting (WB) was performed with anti-FLAG antibody. B, templates bearing an alanine substitution for each of the C-terminal 18 amino acids of 2A (except two native alanine residues) and for the N-terminal four amino acids of 2B in the plasmid pUC-T7-HCV 2A (18 aa)-2B-FLAG were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-FLAG antibody. Each amino acid is numbered from the C-terminal amino acid (glycine) of 2A, which is set to +1. The efficiency of processing (2B-FLAG/2B-FLAG + 2A (18 aa)-2B-FLAG) is reported below each panel. C, plasmids pUC-T7-HCV HA-2A-2B-HA (lane 1), pUC-T7-HCV HA-2A(silent-1)2B-HA (lane 2), and pUC-T7-HCV HA-2A(silent-2)2B-HA (lane 3) were incubated in the HeLa cell extract-derived protein synthesis system. Western blotting was performed with anti-HA antibody.
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.

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