Translation of the initial codons of satellite tobacco necrosis virus ribonucleic acid in a cell-free system from wheat embryo.

Satellite tobacco necrosis virus RNA directs the ribosomal binding of methionyl-, alanyl-, and lysyl-tRNA when incubated under the appropriate conditions in the in vitro wheat embryo amino acid incorporating system. The products of the various binding reactions have been identified as methionyl-tRNA, methionyl-alanyl-tRNA, and methionyl-alanyllysyl-tRNA. This sequence corresponds to the known NH2terminal sequence of the satellite tobacco necrosis virus coat protein thereby establishing the accurate initiation and translation of at least the three initial codons of the translated portion of the vira1 RNA. Methionyl-tRNA binding requires, in addition to ATP and GTP, the presence of the two initiation factors C and D, and occurs readily at 1.3 u Mg2+. Alanyl-tRNA binding is obtained only when the system is supplemented with phosphoenolpyruvate, pyruvate kinase, and elongation factor 1 and when the Mg2+ concentration is raised to 3.6 11~1. These latter conditions also allow an additional increment of methionyl-tRNA to be bound to ribosomes. The amount of extra methionyl-tRNA bound is equal to the alanyl-tRNA bound under these conditions and to the methionyl-tRNA bound in the original initiation reaction. Chlortetracycline completely inhibits binding of alanyl-tRNA as well as the extra increment of methionyl-tRNA. It is, however, without effect on the initial binding of methionyl-tRNA. Binding of lysyl-tRNA to ribosomes occurs only upon further supplementation with elongation factor 2. These observations provide further evidence that factors C and D are initiation factors; that elongation factor 1 functions in dipeptide synthesis and that elongation factor 2 is required only for tripeptide formation. The ability to bind the extra increment of methionyl-tRNA under conditions of dipeptidyl tRNA synthesis suggests the existence of a third ribosomal binding site specific for methionyl-tRNA.