Translation of Satellite Tobacco Necrosis Virus Ribonucleic Acid. II. Initiation of in Vitro Translation in Procaryotic and Eucaryotic Systems

This paper reports the N-terminal amino acid of the product of in vitro translation of satellite tobacco necrosis virus ribonucleic acid (STNV-RNA) by both a procaryotic (Escherichia coli) and eucaryotic (wheat embryo) system. In vitro translation of satellite tobacco necrosis virus RNA by the procaryotic (Escherichia coli) system initiates with fMet-tRNA1met. Specifically, deformylation of the in vitro product protein followed by end-group analysis with fluo-rodinitrobenzene reveals DNP-Met. At 6-7 mM Mg2+ levels, extracts from Escherichia coli deprived of formyl donors by the action of trimethoprim require formyltetrahydrofolic acid for translation of the RNA. The viral-RNA-dependent incorporation of [3H]formate from [3H]formyltetrahydro- folic acid into protein results in the preferential labeling of one tryptic fingerprint peptide. Digestion of the in vitro product protein with specific proteases followed by ion-exchange procedures yields N-formylmethionine. Similar ion-exchange analyses of the product of in vitro translation of STNV-RNA by the eucaryotic (wheat embryo) system fail to detect fMet in the product. In contrast, end-group analyses of the in vitro eucaryotic product reveal Ala as the most prevalent N-terminal amino acid. These data support the theory that the original, eucaryotic, STNV-RNA translation product has specifically lost an N-terminal methionine to yield an alanine-terminated protein.