title: 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.
abstract: The genomic RNA of encephalomyocarditis virus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins 2A and 2B by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3 (eRF1 and eRF3), aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site. 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.
authors.affiliation: Department of Materials Science and Chemistry and University of Hyogo, Himeji 671-2201, Japan; Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan and.
authors.forename: Kodai
authors.initials: K
authors.lastname: Machida
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mikami
authors.affiliation: Department of Materials Science and Chemistry and University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Mamiko
authors.initials: M
authors.lastname: Masutani
authors.affiliation: Department of Materials Science and Chemistry and University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Kurumi
authors.initials: K
authors.lastname: Mishima
authors.affiliation: Department of Materials Science and Chemistry and University of Hyogo, Himeji 671-2201, Japan.
authors.forename: Tominari
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Materials Science and Chemistry and University of Hyogo, Himeji 671-2201, Japan; Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan and. Electronic address: imataka@eng.u-hyogo.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Imataka
mesh_terms: D000409:Alanine / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001665:Binding Sites; D002474:Cell-Free System; D018076:DNA, Complementary / Q000378:metabolism; D004686:Encephalomyocarditis virus / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006367:HeLa Cells; D016174:Hepacivirus / Q000378:metabolism; D006801:Humans; D009154:Mutation; D016366:Open Reading Frames; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D017510:Protein Folding; D012270:Ribosomes / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D015852:Eukaryotic Initiation Factor-2; D020648:Peptide Elongation Factor 1; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C057421:virus protein 2A; D000604:Amino Acyl-tRNA Synthetases; D000409:Alanine
keywords: Cell-free System; Protein Expression; Protein Processing; Protein Synthesis; Reconstitution System; Translation Regulation; Viral Protein