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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1972 Dec;69(12):3589–3593. doi: 10.1073/pnas.69.12.3589

Initiation of Picornavirus Protein Synthesis in Ascites Cell Extracts

Bo F Öberg 1,*, Aaron J Shatkin 1
PMCID: PMC389827  PMID: 4345505

Abstract

The current model of picornavirus protein formation implies that initiation of protein synthesis occurs at a single site on the viral RNA, and that the large polypeptide formed is later cleaved. A direct test of this model was made in vitro by studying the incorporation of [35S]methionine from rabbit liver Met-tRNAMMet and fMet-tRNAFMet into encephalomyocarditis virus RNA-coded proteins in extracts of Ehrlich ascites cells. The incorporation of N-formylmethionine was complete within 5 min, while utilization of Met-tRNAMMet continued for 20 min. Tryptic digests of [35S]methionine-labeled products from Met-tRNAMMet analyzed by anion-exchange chromatography yielded more than 30 peptides, as compared to about 15 [35S]methionine-labeled peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-tRNAFMet yielded one major 26S-labeled tryptic peptide. The N-terminal location of methionine in this peptide was verified by Edman degradation. One predominant N-terminal tryptic peptide was also obtained with fMet-tRNAFMet when mouse Elberfeld and mengo-virus RNAs were used as messengers. On the basis of N-terminal compared with internal labeling of the products, no evidence for in vitro post-translational cleavage was found. The results are consistent with a single initiation site for synthesis of picornavirus proteins.

Keywords: translation in vitro, Met-tRNA, tryptic mapping, encephalomyocarditis RNA

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Selected References

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