The Initiation of Protein Synthesis Directed by the RNA from Encephalomyocarditis Virus

Abstract

The initiation of protein synthesis directed by the RNA from encephalomyocarditis virus in a cell‐free system from Krebs II ascites cells has been investigated. Ascites cell Met‐tRNAMet donates all the internal methionyl residues of the viral‐RNA‐specified polypeptide, whereas little incorporation from Met‐tRNAfMet is detected. fMet‐tRNAfMet, which is not normally present in the cytoplasm of eukaryotic cells, donates formylmethionine into polypeptide in response to the viral RNA to give a product which contains a single N‐terminal sequence fMet‐Ala‐Thr‐, and is identical to the normal cell‐free polypeptide except for the N‐terminal formylmethionyl residue. Short‐chain peptidyl‐tRNA synthesised in vitro in response to encephalomyocarditis virus RNA in the presence of low concentrations of sparsomycin, which inhibits chain elongation but not initiation, contains the N‐terminal sequence Met‐Ala‐Thr‐. This data supports the notion that unblocked Met‐tRNAfMet is the universal initiator of protein synthesis in eukaryotes and donates a methionyl residue into the N‐terminal position of all proteins. The initiating methionyl residue is in most cases subsequently removed from the nascent polypeptide, but this can be inhibited either by an N‐terminal formyl group or by preventing the nascent polypeptide from growing long enough to be a substrate for the methionine aminopeptidase. The data also shows that encephalomyocarditis virus RNA, which codes for at least nine stable proteins in vivo, contains only one initiation site which is expressed in vitro. The polypeptide coded for immediately after the initiation site is not virus‐coat protein and is rapidly metabolised in vivo, suggesting that a lead‐in polypeptide precedes the first meaningful protein sequence. Copyright © 1973, Wiley Blackwell. All rights reserved