DISCRIMINATION BETWEEN INITIATION AND ELONGATION OF PROTEIN-BIOSYNTHESIS IN YEAST - IDENTITY ASSURED BY A NUCLEOTIDE MODIFICATION IN THE INITIATOR TRANSFER-RNA

Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(i)Met, this modified nucleoside located in the T-stem of the tRNA is a 2'-1''-(beta-O-ribofuranosyl-5''-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(i)Met and regeneration of the 3'-terminal adenosine with ATP(CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(i)Met to eEF-1alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1alpha, thus preventing the initiator tRNA(i)Met from entering the elongation cycle of protein biosynthesis.