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. 1972 May;69(5):1234–1238. doi: 10.1073/pnas.69.5.1234

Formation of a Mammalian Initiation Complex with Reovirus Messenger RNA, Methionyl-tRNAF, and Ribosomal Subunits

Daniel H Levin 1, David Kyner 1, George Acs 1
PMCID: PMC426671  PMID: 4504335

Abstract

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [36S]Met-tRNAF and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [35S]Met-tRNAF; rat liver [35S]Met-tRNAM was unreactive unless the supernatant factors EF T1 and EF T2 were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [35S]Met-tRNAF, completely inhibited Met-tRNAM utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNAF at a puromycin-reactive site on ribosomes.

Keywords: protein initiation, binding, puromycin peptides, Met-tRNAM

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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