RRC ID 33252
Author Machida K, Mikami S, Masutani M, Mishima K, Kobayashi T, Imataka H.
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.
Journal J Biol Chem
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.
Volume 289(46)
Pages 31960-71
Published 2014-11-14
DOI 10.1074/jbc.M114.593343
PII M114.593343
PMID 25258322
PMC PMC4231674
MeSH Alanine / genetics Amino Acyl-tRNA Synthetases / metabolism Binding Sites Cell-Free System DNA, Complementary / metabolism Encephalomyocarditis virus / metabolism* Eukaryotic Initiation Factor-2 / metabolism* Gene Deletion Gene Expression Regulation HeLa Cells Hepacivirus / metabolism Humans Mutation Open Reading Frames Peptide Elongation Factor 1 / metabolism* Plasmids / metabolism Protein Biosynthesis* Protein Folding Ribosomes / chemistry Viral Nonstructural Proteins / chemistry* Viral Nonstructural Proteins / metabolism* Viral Proteins / metabolism*
IF 4.106
Times Cited 13
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pUC-T7-HCV HA-2A-2B-FLAG (RDB13261) pUC-T7-HCV HA-2A-2B-HA (RDB13262) pUC-T7-HCV HA-2A(silent-1)2B-HA (RDB13263) pUC-T7-HCV HA-2A(silent-2)2B-HA (RDB13264) pUC-T7-HCV HA-2A(uORF2) (RDB13265) pUC-T7-EMCV-His-eEF1A (RDB13266) pUC-T7-EMCV-His-eEF1Bgamma (RDB13267) pUC-T7-EMCV-eEF1Balpha (RDB13268) pGEX6P-eRF1 (RDB13269) pGEX6P-eRF3 (RDB13270) pUC-T7-EMCV-GST-Glu-ProRS (RDB13271) pUC-T7-EMCV-GST-MetRS (RDB13272) pUC-T7-EMCV-GST-AspRS (RDB13273) pUC-T7-EMCV-GST-AlaRS (RDB13274) pUC-T7-EMCV-GST-CysRS (RDB13275) pUC-T7-EMCV-GST-ValRS (RDB13276) pUC-T7-EMCV-GST-TyrRS (RDB13277) pUC-T7-EMCV-His-PheRSbeta (RDB13278) pUC-T7-HCV-Rluc-HA (RDB13279) pUC-T7-HCV-beta-actin-HA (RDB13280) pUC-T7-HCV-PABP-HA (RDB13281) pUC-T7-HCV-beta-Gal-HA (RDB13282) pUC-T7-HCV-HA-Rluc (RDB13283) pUC-T7-HCV-HA-beta-actin (RDB13284) pUC-T7-HCV-HA-PABP (RDB13285) pUC-T7-HCV-HA-beta-Gal (RDB13286) pUC-T7-HCV HA-2A-HA-2B (22 aa) (RDB13287).