Tobacco Mosaic Virus Ribonucleic Acid-dependent Amino Acid Incorporation in a Wheat Embryo System in Vitro

Abstract Kinetics of tobacco mosaic virus (TMV)-RNA-mediated amino acid incorporation into peptide in a wheat embryo system showed a 5- to 8-min lag. In comparative experiments with polyribosomes (in which the messenger and ribosomes are already attached) and polyuridylic acid (in which the ribosome-messenger attachment is nonspecific), amino acid incorporation was linear from zero time. When peptide synthesis was followed by aminoacyl transfer from aminoacyl-tRNA, the kinetic lag in the TMV-RNA system was retained, indicating that the synthesis of aminoacyl-tRNA is not the rate-limiting reaction. If, however, TMV-RNA and ribosomes were previously incubated in the presence of ATP and two supernatant components, the lag was removed. These observations suggest that the rate-limiting reaction in the TMV-RNA system is the formation of a ribosome-messenger initiation complex and that ATP and two soluble factors are involved in its formation. The ATP requirement in the TMV-RNA system was completely retained when peptide synthesis was studied by aminoacyl transfer from aminoacyl-tRNA. In contrast, in similar aminoacyl transfer experiments with polyribosomes and polyuridylic acid, only GTP was required and ATP was without effect. These results indicate that ATP is functioning in the TMV-RNA system in some capacity other than to provide aminoacyl-tRNA. When previously incubated mixtures of the TMV-RNA system were passed through Sephadex G-25, the excluded fraction catalyzed aminoacyl transfer in the absence of ATP, suggesting that the product formed during preliminary incubation was a macromolecular complex. Analysis of the requirements for formation of this active complex supports the contention that it is a ribosome-messenger complex. Similar experiments, in which puromycin or cycloheximide were included in the preliminary incubation and then removed by Sephadex G-25 filtration, suggest that aminoacyl transfer is not obligatory to the formation of the complex.