GENE-SPECIFIC TRANSLATIONAL CONTROL OF THE YEAST GCN4 GENE BY PHOSPHORYLATION OF EUKARYOTIC INITIATION FACTOR-II

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) is one of the best-characterized mechanisms for down-reg ulating total protein synthesis in mammalian cells in response to vari ous stress conditions. Recent work indicates that regulation of the GC N4 gene of Saccharomyces cerevisiae by amino acid availability represe nts a gene-specific case of translational control by phosphorylation o f eIF-2alpha. Four short open reading frames in the leader of GCN4 mRN A (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes t ranslate the first uORF and reinitiate at one of the remaining uORFs i n the leader, after which they dissociate from the mRNA. Under conditi ons of amino acid starvation, many ribosomes which have translated uOR F1 fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon in stead. Failure to reinitiate at uORFs 2-4 in starved cells results fro m a reduction in the GTP-bound form of eIF-2 that delivers charged ini tiator tRNA(i)Met to the ribosome. When the levels of eIF-2.GTP.Met-tR NA(i)Met ternary complexes are low, many ribosomes will not rebind thi s critical initiation factor following translation of uORF1 until afte r scanning past uORF4, but before reaching GCN4. Phosphorylation of eI F-2 by the protein kinase GCN2 decreases the concentration of eIF-2.GT P.Met-tRNA(i)Met complexes by inhibiting the guanine nucleotide exchan ge factor for eIF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of eIF-2.