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

Journal Article · Fri Oct 01 04:00:00 EDT 1976 · Virology; (United States)

DOI:https://doi.org/10.1016/0042-6822(76)90346-9· OSTI ID:7314579

Samuel, C E ^[1]; Joklik, W K

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1. Univ. of California, Santa Barbara

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 ..mu../sub 0/, ..mu../sub 1/, sigma/sub 2a/, and sigma/sub 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 ..mu../sub 0/, ..mu../sub 1/, sigma/sub 2a/, and sigma/sub 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-Formylmethionyl-containing 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: ..mu../sub 0/, (N-formyl)methionyl-valyl-(proline); ..mu../sub 1/, (N-formyl)methionyl-leucyl-valine; sigma/sub 2a/, (N-formyl)methionyl-threonyl-valine; and sigma/sub 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.

• OSTI ID: 7314579
• Journal Information: Virology; (United States), Journal Name: Virology; (United States) Vol. 74:2; ISSN VIRLA
• Country of Publication: United States
• Language: English

Related Subjects

550700* -- Microbiology
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BIOSYNTHESIS
CARBOXYLIC ACIDS
IN VITRO
MESSENGER-RNA
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PROTEINS
RNA
SYNTHESIS
TRANSFER RNA
VIRUSES