Two initiation sites detected in the small s1 species of reovirus mRNA by dipeptide synthesis in vitro.

Reovirus mRNAs directed the synthesis of fMet dipeptides in a translation initiation system reconstituted from rabbit reticulocyte initiation and elongation factors, Artemia salina 80S ribosomes, yeast fMet-tRNAiMet and Escherichia coli3H-labeled aminoacyl tRNAs. As predicted from the GC(U,G) codon that follows the 5'-proximal AUG in half of the viral mRNA species, fMet-Ala was the predominant dipeptide product obtained in response to a mixture of mRNAs or to the separated size classes of medium (m) and small (s) mRNA. The four individual small mRNA species each directed the synthesis of an fMet dipeptide that was consistent with the utilization of the 5'-proximal AUG for initiation. In addition to fMet-Asp, the s1 mRNA also directed fMet-Glu synthesis indicative of initiation in a second reading frame at the 5'-penultimate AUG. The tripeptide fMet-Glu-Tyr was also synthesized from s1 mRNA, which further verified this second initiation site. mRNAs containing 5'-terminal GpppG were 10-15% as active as the corresponding m7G-capped templates. The dipeptide assay provides a rapid method for determining initiation sites in individual mRNAs or in mixtures of mRNAs.