MULTICOPY TRANSFER-RNA GENES FUNCTIONALLY SUPPRESS MUTATIONS IN YEASTEIF-2-ALPHA KINASE GCN2 - EVIDENCE FOR SEPARATE PATHWAYS COUPLING GCN4 EXPRESSION TO UNCHARGED TRANSFER-RNA

GCN2 is a protein kinase that stimulates translation of GCN4 mRNA in a mino acid-starved cells by phosphorylating the alpha subunit of transl ation initiation factor 2 (eIF-2). We isolated multicopy plasmids that overcome the defective derepression of GCN4 and its target genes caus ed by the leaky mutation gcn2-507. One class of plasmids contained tRN A(His) genes and conferred efficient suppression only when cells were starved for histidine; these plasmids suppressed a gcn2 deletion much less efficiently than they suppressed gcn2-507. This finding indicates that the reduction in GCN4 expression caused by gcn2-507 can be overc ome by elevating tRNA(His) expression under conditions in which the ex cess tRNA cannot be fully aminoacylated. The second class of suppresso r plasmids all carried the same gene encoding a mutant form of tRNA(Va l)(AAC) with an A-to-G transition at the 3' encoded nucleotide, a muta tion shown previously to reduce aminoacylation of tRNA(Val) in vitro. In contrast to the wild-type tRNA(His) genes, the mutant tRNA(Val) gen e efficiently suppressed a gcn2 deletion, and this suppression was ind ependent of the phosphorylation site on eIF-2 alpha (Ser-51). Overexpr ession of the mutant tRNA(Val) did, however, stimulate GCN4 expression at the translational level. We propose that the multicopy mutant tRNA (Val) construct leads to an accumulation of uncharged tRNA(Val) that d erepresses GCN4 translation through a pathway that does not involve GC N2 or eIF-2 alpha phosphorylation. This GCN2-independent pathway was a lso stimulated to a lesser extent by the multicopy tRNA(His) construct s in histidine-deprived cells. Because the mutant tRNA(Val) exacerbate d the slow-growth phenotype associated with eIF-2 alpha hyperphosphory lation by an activated GCN2(c) kinase, me suggest that the GCN2-indepe ndent derepression mechanism involves down-regulation of eIF-2 activit y.