Biosynthesis of reovirus-specified polypeptides. Initiation of reovirus messenger RNA translation in vitro and identification of methionyl-x initiation peptides.

Abstract

Abstract The initiation of reovirus messenger RNA-directed protein synthesis in vitro was investigated in a cell-free protein synthesizing system prepared from Krebs ascites tumor cells. The principal translation products of the mixture of 10 reovirus mRNA species transcribed in vitro by reovirus cores were polypeptides μ 0 , μ 1 , σ 2a , and σ 3 . Translation could be initiated with formylmethionine transferred from rat liver methionyl-tRNA formylated by Escherichia coli formyltransferase with 10-formyltetrahydrafolate as the formyl donor. Formylmethionine incorporation was complete within 10 to 15 min and was inhibited by aurin tricarboxylic acid and pactamycin; by contrast, incorporation of methionine and leucine continued for 30 to 60 min. The identification of the amino acids at the amino termini of polypeptides μ 0 , μ 1 , σ 2a , and σ 3 synthesized in vitro was elucidated. Protein synthesis was carried out in the presence of rat liver formylmethionyl-tRNA and various groups of radioactively labeled amino acids. The viral polypeptides that were synthesized were isolated by urea-SDS-polyacrylamide gel electrophoresis and digested with pronase. N -Formylmethionylcontaining peptides were then separated from other peptides by fractionation on Dowex-50 and hydrolyzed with acid. The radioactive amino acids that were liberated were then identified by two-dimensional thin-layer chromatography. The following amino terminal assignments were elucidated: μ 0 , ( N -formyl)methionyl-valyl-(proline); μ1, ( N -formyl)methionyl-leucyl-valine; σ 2a , ( N -formyl)methionyl-threonyl-valine; and σ 3 , ( N -formyl)methionyl-valyl-tyrosyl-(proline). No evidence was obtained for amino terminal acetylation or formylation of reovirus-specific protein synthesized in vitro in the absence of exogenously added formylmethionyl-transfer RNA.