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 ability to synthesize reovirus messenger RNA (mRNA) in vitro in quantity (1-4) is useful for a study of the mechanism of protein initiation in mammalian systems. In eukaryotic cells there exist two major methionyl-tRNA species (5-10) that have been identified, by several criteria, as MettRNAF and Met-tENAx. As for the bacterial initiator tRNA, eukarytic Met-tRNAF is an initiator of protein synthesis in eukaryotic cell-free systems (8, 11-18) and, in contrast to Met-tRNAm, can be acylated and formylated by bacterial enzymes (7,8,13,(16)(17)(18)(19)(20) In the studies described here, we observed that purified rat liver Met-tRNAF, but not rat liver Met-tRNAm, formed an initiation complex with ribosomal subunits that was dependent upon the presence of reovirus mRNA. The mRNAdependent complex reacted with puromycin to form methionyl-puromycin in the same manner as the initiation complexes formed in the presence of poly(A,G,U) or the initiator triplet AUG.
METHODSThe growth of mouse fibroblast L-929 cells, infection by reovirus (Dearing strain, Type 3), the preparation of reovirus RNA transcriptase, and the synthesis in vitro of reovirus mRNA have been described (4).Ribosomal Subunits from L-929 Cells. All operations were performed at 0-40, unless otherwise indicated. In a standard preparation, 109 L-929 cells were harvested during exponential growth, and extracts were prepared by homogenization in 15 ml of 10 mM Tris HCl (pH 7.5)-10 mM KCl-1.5 mM Mg-C12-1 mM dithiothreitol as described (4). The extract was brought to 30 mM Tris HOl (pH 7.5)-130 mM KCI-6 mM MgCl2-3 mM dithiothreitol (TKM buffer). The mitochondria were removed by centrifugation. The extract was supplemented with 20 amino acids, ATP, GTP, creatine phosphate, phosphocreatine kinase, and 1 mM puromycin, and incubated at 370 for 50 min. The reaction mixture was made 0.8% in deoxycholate and centrifuged overnight at 160,000 X g through a cushion of 68% sucrose in TKM buffer. The pellet was suspended in 3 ml of 50 mM triethanolamine-HCl (pH 7.6)-500 mM KCl-1.5 mM MgCl2-1 mM puromycin, incubated at 00 for 10 min and at 370 for 10 min, and then centrifuged at 200 in 10-30% sucrose gradients in 50 mM triethanolamine-HCl (pH 7.6)-500 mM KCI-5 mM MgCl2 for 5.5 hr at 64,000 X g (22). The gradients were monitored for A20, and the 40S and 60S subunit areas were separately collected and centrifuged at 160,000 X g overnight through a cushion of 40% sucrose in TKM buffer. The subunit pellets were each resuspended in TKM buffer containing 20% glycerol and could be stored at 00 for 2 weeks without significant loss of activity.Preparation of Rat Liver [*S1]Met-tRNAF and [n5S]MettRNAm. Rat liver tRNA (110 mg) was fractionated by chromatography on BD-cellulose, as described for wheat germ tRNA by Leis and Keller (9). The location of the two major tRNAMet peaks in the A20 profile ...