MODULATION OF TRNA(I)(MET), EIF-2, AND EIF-2B EXPRESSION SHOWS THAT GCN4 TRANSLATION IS INVERSELY COUPLED TO THE LEVEL OF EIF-2-CENTER-DOT-GTP-CENTER-DOT-MET-TRNA(I)(MET) TERNARY COMPLEXES

To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation ini tiation, we examined the effects of altering the gene dosage of initia tor tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for e IF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all fiv e subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosph orylation on both GCN4-specific and general translation initiation. Co nsistent with eIF-2 functioning in translation as part of a ternary co mplex composed of eIF-2, GTP, and Met-tRNA(i)(Met), reduced gene dosag e of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and s timulated GCN4 translation. In addition, overexpression of a combinati on of eIF-2 and tRNA(i)(Met) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2 . GTP . Met -tRNA(i)(Met) ternary complex is the principal component limiting tran slation in cells when eIF-2 alpha is phosphorylated on serine 51. Anal ysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a co mpetitive inhibitor of eIF-2B rather than forming an excessively stabl e inactive complex. Finally, our results demonstrate that the concentr ation of eIF-2 . GTP . Met-tRNA(i)(Met) ternary complexes is the cardi nal parameter determining the site of reinitiation on GCN4 mRNA and su pport the idea that reinitiation at GCN4 is inversely related to the c oncentration of ternary complexes in the cell.