GCN4 is a transcriptional activator of amino acid biosynthetic genes that is itself regulated at the translational level by short open reading frames (uORFs) present in the leader of GCN4 mRNA. uORFs 3 and 4 repress GCN4 expression in nonstarvation conditions, whereas UORF1 stimulates GCN4 expression by overcoming the inhibitory effects of uORFs 3 and 4. The distinct functions of uORFs 1 and 4 are determined by sequences immediately surrounding their stop codons, suggesting that translation termination occurs differently at these two sites. Reinitiation after uORF4 translation is very inefficient in nonstarvation conditions; by contrast, UORF1 translation is compatible with downstream initiation and this property is important for its ability to regulate uORF4. We propose that translation of UORF1 alters the ribosome in a way that facilitates its advance through uORF4 sequences in amino acid-starved cells. Positive and negative transacting factors are also required to regulate ribosome movement in the GCN4 mRNA leader. Modulating the activity of the general translation initiation factor eIF-2 appears to be one important function of the positive regulators GCN2 and GCN3. At least two negative regulators of GCN4 expression, GCD1 and GCD2, are essential factors that probably also function in translation initiation. Interestingly, GCD1 and GCD2 appear to exist in a complex with GCN3, suggesting that GCN3 directly modulates the activity of these gene products. GCN2 stimulates GCN4 expression by functioning as a protein kinase. Homology between the C-terminus of GCN2 and histidyl-tRNA synthetases suggests that GCN2 detects amino acid starvation by directly monitoring the concentration of uncharged tRNA. In this model, tRNA binding stimulates GCN2 kinase activity. Mutations in GCD genes, in genes encoding subunits of eIF-2, and in GCN3 have been identified that all bypass the requirement for GCN2 to derepress GCN4 expression, making these factors potential substrates for phosphorylation by GCN2 in amino acid-starved cells. Ribosomal protein (rp) genes are repressed under the same amino acid starvation conditions that lead to elevated expression of amino acid biosynthetic genes under GCN4 control. Repression of the RPL16A rp gene requires regulatory sequences that include the binding site for the transcriptional activator TUF. Regulation of RPL16A expression occurs independently of GCN factors, indicating that separate pathways exist for these two transcriptional responses to amino acid starvation.
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