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

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Abstract
Reovirus mRNA directed the synthesis of fMet dipeptides in a translation initiation system reconstituted from rabbit reticulocyte initiation and elongation factors, Artemia salina 80S ribosomes, yeast .**GRAPHIC**. and Escherichia coli 3H-labeled aminoacyl tRNA. 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 mRNA or to the separated size classes of medium (m) and small (s) mRNA. The 4 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 2nd reading frame at the 5''-penultimate AUG. The tripeptide fMet-Glu-Tyr was also synthesized from s1 mRNA, which further verified this 2nd initiation site. mRNAs containing 5''-termination 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 mRNA or in mixture of mRNA.