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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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Purification of each component used in the reconstituted protein synthesis system.A, ribosomes. 40 and 60 S ribosomal subunits were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue (CBB) (left two panels) or resolved on a denatured agarose gel (1%) and stained with ethidium bromide (right two panels). B, tRNAs. tRNAs (2 μg) were resolved by 8 m urea PAGE (12.5%) and stained with toluidine blue (TB). C, translation factors. eEF1s (eEF1A, eEF1Bα, and eEF1Bγ), eEF2, eRF1, and eRF3 (1.5 μg each) were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue. D, left panel, AlaRS, CysRS, PheRS (α + β), GlyRS, HisRS, AsnRS, SerRS, ThrRS, ValRS, TrpRS, and TyrRS (1 μg each). Right panel, a mixture of Glu-ProRS, IleRS, LeuRS, MetRS, GlnRS, ArgRS, LysRS, and AspRS (8 μg) resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue.
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Figure 4: Purification of each component used in the reconstituted protein synthesis system.A, ribosomes. 40 and 60 S ribosomal subunits were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue (CBB) (left two panels) or resolved on a denatured agarose gel (1%) and stained with ethidium bromide (right two panels). B, tRNAs. tRNAs (2 μg) were resolved by 8 m urea PAGE (12.5%) and stained with toluidine blue (TB). C, translation factors. eEF1s (eEF1A, eEF1Bα, and eEF1Bγ), eEF2, eRF1, and eRF3 (1.5 μg each) were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue. D, left panel, AlaRS, CysRS, PheRS (α + β), GlyRS, HisRS, AsnRS, SerRS, ThrRS, ValRS, TrpRS, and TyrRS (1 μg each). Right panel, a mixture of Glu-ProRS, IleRS, LeuRS, MetRS, GlnRS, ArgRS, LysRS, and AspRS (8 μg) resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue.

Mentions: Experiments in yeast genetics have suggested that eRFs are involved in the translation termination of 2A (19). To examine whether eRFs were required for 2A-2B processing in a mammalian system, we established a protein synthesis system reconstituted with purified eukaryotic factors. To simplify the system, we used the IRES of HCV to promote translation initiation without eIFs; a previous study had shown that the 80 S ribosome bound to the HCV IRES without eIFs at a relatively high concentration of magnesium (13). We expressed recombinant human eEF1s (eEF1A, eEF1Bγ, and eEF1Bα) and ARSs in BHK-21 cells, and we expressed eRF1 and eRF3 in bacteria. These components were then purified (Fig. 4). Endogenous eEF2, the 40 and 60 S ribosomal subunits, and total tRNAs were purified from HeLa cells (Fig. 4). These components were combined in a test tube with amino acids, triphosphate nucleotides (ATP, GTP, CTP, and UTP), and T7 RNA polymerase. The concentration of magnesium was set to 6.0 mm, but the concentration of free magnesium in the system was unknown because the triphosphate nucleotides chelate magnesium (the total nucleotide concentration was 3.7 mm: 1.25 mm ATP and 0.83 mm each of GTP, CTP, and UTP).


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)

Purification of each component used in the reconstituted protein synthesis system.A, ribosomes. 40 and 60 S ribosomal subunits were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue (CBB) (left two panels) or resolved on a denatured agarose gel (1%) and stained with ethidium bromide (right two panels). B, tRNAs. tRNAs (2 μg) were resolved by 8 m urea PAGE (12.5%) and stained with toluidine blue (TB). C, translation factors. eEF1s (eEF1A, eEF1Bα, and eEF1Bγ), eEF2, eRF1, and eRF3 (1.5 μg each) were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue. D, left panel, AlaRS, CysRS, PheRS (α + β), GlyRS, HisRS, AsnRS, SerRS, ThrRS, ValRS, TrpRS, and TyrRS (1 μg each). Right panel, a mixture of Glu-ProRS, IleRS, LeuRS, MetRS, GlnRS, ArgRS, LysRS, and AspRS (8 μg) resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231674&req=5

Figure 4: Purification of each component used in the reconstituted protein synthesis system.A, ribosomes. 40 and 60 S ribosomal subunits were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue (CBB) (left two panels) or resolved on a denatured agarose gel (1%) and stained with ethidium bromide (right two panels). B, tRNAs. tRNAs (2 μg) were resolved by 8 m urea PAGE (12.5%) and stained with toluidine blue (TB). C, translation factors. eEF1s (eEF1A, eEF1Bα, and eEF1Bγ), eEF2, eRF1, and eRF3 (1.5 μg each) were resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue. D, left panel, AlaRS, CysRS, PheRS (α + β), GlyRS, HisRS, AsnRS, SerRS, ThrRS, ValRS, TrpRS, and TyrRS (1 μg each). Right panel, a mixture of Glu-ProRS, IleRS, LeuRS, MetRS, GlnRS, ArgRS, LysRS, and AspRS (8 μg) resolved by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue.
Mentions: Experiments in yeast genetics have suggested that eRFs are involved in the translation termination of 2A (19). To examine whether eRFs were required for 2A-2B processing in a mammalian system, we established a protein synthesis system reconstituted with purified eukaryotic factors. To simplify the system, we used the IRES of HCV to promote translation initiation without eIFs; a previous study had shown that the 80 S ribosome bound to the HCV IRES without eIFs at a relatively high concentration of magnesium (13). We expressed recombinant human eEF1s (eEF1A, eEF1Bγ, and eEF1Bα) and ARSs in BHK-21 cells, and we expressed eRF1 and eRF3 in bacteria. These components were then purified (Fig. 4). Endogenous eEF2, the 40 and 60 S ribosomal subunits, and total tRNAs were purified from HeLa cells (Fig. 4). These components were combined in a test tube with amino acids, triphosphate nucleotides (ATP, GTP, CTP, and UTP), and T7 RNA polymerase. The concentration of magnesium was set to 6.0 mm, but the concentration of free magnesium in the system was unknown because the triphosphate nucleotides chelate magnesium (the total nucleotide concentration was 3.7 mm: 1.25 mm ATP and 0.83 mm each of GTP, CTP, and UTP).

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