GCN4 is a transcriptional activator of amino acid biosynthetic genes in S. cerevisiae that is regulated at the translational level. Upstream open reading frames (uORFs) in its mRNA leader regulate the flow of scanning ribosomes to the GCN4 start codon according to the availability of amino acids. Our mutational analysis of GCN4 strongly suggests that ribosomes which translate GCN4 have previously translated uORF1, resumed scanning and bypassed uORFs 2-4. This occurs under starvation conditions because reinitiation of translation is inefficient under these circumstances. GCD factors are required for translational repression of GCN4. GCD1 and GCD2 have general functions in translation initiation and exist in a high molecular weight complex with a fraction of initiation factor 2 (elF-2) present in the cell. GCD6 and GCD7 proteins also appear to be components or regulators of this complex. In addition, mutations in elF-2 subunits have a Gcd phenotype. These and other findings have led us to suggest that the high molecular weight GCD protein complex is the yeast equivalent of elF-2B, a factor that recycles eIF-2 following each round of translation initiation. elF-2B activity is down-regulated in mammalian cells by phosphorylation of the alpha subunit of elF-2. Interestingly, the yeast GCN2 protein kinase Is predicted to be an antagonist of GCD factors and exhibits a high level of sequence similarity to the mammalian elF-2alpha kinase called DAI. Like DAI, GCN2 is a ribosome-associated protein. Recently, we have shown that GCN2 can phosphorylate in vitro elF-2alpha isolated from rabbit or yeast and that mutation of the predicted phosphorylation site on yeast elF-2alpha impairs translational derepression of GCN4. These results suggest that regulation of GCN4 expression represents a gene-specific example of the modulation of elF-2 recycling by phosphorylation. Ribosomal protein (rp) genes are transcriptionally repressed under amino acid starvation conditions. This repression is mediated by the binding site for the regulatory protein RAP1. RAP1 activation of rp genes is modulated by amino acid levels at a post- translational level, by a mechanism independent of GCN1-GCN4. Thus, separate pathways exist for regulating amino acid biosynthesis and ribosome biogenesis in response to amino acid availability.
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