Protein Chain Initiation by Methionyl-tRNA in Wheat Embryo

The mechanism of protein chain initiation has been investigated in a cell-free amino acid incorporation system from wheat embryos dependent on tobacco mosaic virus RNA. Analysis of the N-termini of the labeled peptide products of short-term incubations showed the presence of unblocked methionine. In addition, methionyl-tRNA (Met-tRNA) could be bound to ribosomes at 1.3 mM Mg(++) in a reaction requiring viral RNA, ATP, GTP, and soluble protein factors. Incorporation experiments with the two cytoplasmic Met-tRNAs of wheat germ, an initiating species designated Met-tRNA(i) and a Met-tRNA(m), showed that methionine transfer from Met-tRNA(i) was linear from zero time, while that from Met-tRNA(m) occurred only after an appreciable lag. Analysis of the peptide products showed that methionine transfer from Met-tRNA(i) was predominantly N-terminal. In contrast, methionine transfer from Met-tRNA(m) was exclusively into internal positions. Similar selectivity was observed in the ribosome binding assay; only Met-tRNA(i) showed a strong reaction. These experiments provide strong evidence that in the wheat embryo, cytoplasmic Met-tRNA(i) functions without formylation in the initiation of protein synthesis.