TRANSLATION OF THE YEAST TRANSCRIPTIONAL ACTIVATOR GCN4 IS STIMULATEDBY PURINE LIMITATION - IMPLICATIONS FOR ACTIVATION OF THE PROTEIN-KINASE GCN2

The transcriptional activator protein GCN4 is responsible for increase d transcription of more than 30 different amino acid biosynthetic gene s in response to starvation for a single amino acid. This induction de pends on increased expression of GCN4 at the translational level. We s how that starvation for purines also stimulates GCN4 translation by th e same mechanism that operates in amino acid-starved cells, being depe ndent on short upstream open reading frames in the GCN4 mRNA leader, t he phosphorylation site in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), the protein kinase GCN2, and transl ational activators of GCN4 encoded by GCN1 and GCN3. Biochemical exper iments show that eIF-2alpha is phosphorylated in response to purine st arvation and that this reaction is completely dependent on GCN2. As ex pected, derepression of GCN4 in purine-starved cells leads to a substa ntial increase in HIS4 expression, one of the targets of GCN4 transcri ptional activation. gcn mutants that are defective for derepression of amino acid biosynthetic enzymes also exhibit sensitivity to inhibitor s of purine biosynthesis, suggesting that derepression of GCN4 is requ ired for maximal expression of one or more purine biosynthetic genes u nder conditions of purine limitation. Analysis of mRNAs produced from the ADE4, ADE5,7, ADE8, and ADE1 genes indicates that GCN4 stimulates the expression of these genes under conditions of histidine starvation , and it appeared that ADE8 mRNA was also derepressed by GCN4 in purin e-starved cells. Our results indicate that the general control respons e is more global than was previously imagined in terms of the type of nutrient starvation that elicits derepression of GCN4 as well as the r ange of target genes that depend on GCN4 for transcriptional activatio n.