Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4⌬ strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4⌬ mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.In response to environmental perturbations, Saccharomyces cerevisiae cells elicit rapid transcriptional reprogramming involving both activation and repression of gene expression. Transcriptional activator proteins function by binding to specific promoter elements, called upstream activating sequences (UASs) in yeast cells, and recruiting the transcriptional machinery. Thus, transcriptional stimulation requires the expression and function of an activator and the appropriate UAS element in the promoters of its target genes. A plethora of mechanisms are known to regulate the activity or expression of transcriptional activators in response to specific signals. For example, in cells grown on glucose, Gal80p inhibits the ability of Gal4p to activate transcription of genes encoding galactosemetabolizing enzymes, whereas Gal3p alleviates this inhibition on galactose medium (83). The transcriptional activators Pho4p, Swi5p, and Yap1p are regulated by the coupling of their nuclear localization to the levels of inorganic phosphate, cell cycle and mother-daughter status, or oxidative stress, respectively (reviewed in reference 52). Starvation for amino acids, purines, and glucose limitation induces the synthesis of Gcn4p, a bZIP transcriptional activator of amino acid biosynthetic gene...

The GCN2 protein of Saccharomyces cerevisiae stimulates the expression of amino acid biosynthetic genes under conditions of amino acid starvation by derepressing GCN4, a transcriptional activator of these genes. GCN2 contains sequences homologous to the catalytic domain of protein kinases. We show here that substitution of a highly conserved lysine in the presumed ATP-binding site ofthis domain impairs the derepression of histidine biosynthetic genes under GCN4control. This result supports the idea that protein kinase activity is required for GCN2 positive regulatory function.Determination of the nucleotide sequence of the entire GCN2 complementation unit, and measurement of the molecular weight of GCN2 protein expressed in vivo, indicate that GCN2 is a Mr 180,000 protein and contains a Mr 60,000 segment homologous to histidyl-tRNA synthetases (HisRSs) juxtaposed to the protein kinase domain. Several two-codon insertion mutations in the HisRS-related coding sequences inactivate GCN2 regulatory function. Based on these results, we propose that the GCN2 HisRS domain responds to the presence of uncharged tRNA by activating the adjacent protein kinase moiety, thus providing a means of coupling GCN2-mediated derepression of GCN4 expression to the availability of amino acids.Protein phosphorylation is an important posttranslational modification involved in regulating many cellular processes, including signal transduction, growth control, carbon catabolite repression, and protein synthesis (1,2). Protein kinases are often regulated by ligands that bind to regulatory domains or subunits to enhance or inhibit catalytic activity. Examples of this phenomenon are cyclic nucleotide-regulated protein kinases, diacylglycerol activation of protein kinase C, and calmodulin-mediated calcium regulation of phosphorylase kinase and myosin light-chain kinase.A protein kinase has been implicated in the general amino acid control of the yeast Saccharomyces cerevisiae (3). In this system, starvation for any one of at least 10 amino acids, or a defective aminoacyl-tRNA synthetase, leads to increased transcription of 30 or more genes encoding amino acid biosynthetic enzymes in nine different pathways (reviewed in ref. 4). The transcriptional activator GCN4 directly mediates this derepression response. Expression of GCN4 itself is regulated by amino acid availability, but at the level of translation initiation. Trans-acting positive factors encoded by GCNJ, GCN2, and GCN3 are required to stimulate translation of GCN4 mRNA in response to starvation, presumably by antagonism of negative-acting GCD factors (4). A portion of the predicted amino acid sequence of GCN2 is homologous to the catalytic domain of eukaryotic protein kinases and evidence was presented that GCN2 either encodes or regulates a protein that has kinase activity in vitro (3).In this report we show that a highly conserved lysine in the presumptive ATP-binding site of the GCN2 kinase domain is required for derepression of genes under the general control, supporting the...

GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcdl mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORFl from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcdl cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORFl fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORFl translation, causing them all to reinitiate at uORF4 rather than at GCN4.The GCN4 protein of the yeast Saccharomyces cerevisiae is a trahscriptional activator of more than 30 genes involved in the biosynthesis of 10 different amino acids. In response to amino acid starvation, transcription of these genes is stimulated because the rate of GCN4 protein synthesis increases under these conditions. GCN4 expression is regulated by amino acid availability through a translational control mechanism involving four short upstream open reading frames (uORFs) in the leader of GCN4 mRNA. A subset of these uORFs strongly inhibit translation initiation at GCN4 under nonstarvation conditions, and this inhibitory effect is overcome when cells are starved for an amino acid (reviewed in reference 11). Translational repression of GCN4 by the uORFs is dependent on negative regulators encoded by GCD genes. In addition to regulating GCN4 expression, it appears that GCD gene products carry out essential cellular functions, and evidence is accumulating that these functions are involved with the initiation of general protein synthesis (11,35,37). Positive regulators encoded by GCN2 and GCN3 are required for increased translation of GCN4 mRNA under starvation conditions, and these factors are thought to function by antagonizing one or more of the negative-acting GCD proteins (11).Numerous...

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein. We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements. Our results indicate that GCN4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4. One of these domains is coincident with the acidic activation domain defined previously; the other extends over the N-terminal one-third of the protein. Both domains are partially dependent on the coactivator protein ADA2. Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4. At least three of these subdomains are critically dependent on bulky hydrophobic amino acids for their function. Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16. The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved between GCN4 and its Neurospora counterpart, cpc-1. Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inactivate full-length GCN4. These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex.

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the a subunit of translation initiation factor 2 (eIF-2a) in amino acid-starved cells. Phosphorylation of eIF-2a reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN27 alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2' alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class H aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2C alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2c for their effects on GCN4 expression and to increase the level of eIF-2a phosphorylation in the absence of amino acid starvation. Synthetic GCN2" double mutations show greater derepression ofGCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotpes of the GCN2C alleles are dependent on GCNI and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2am kinase GCN2.Phosphorylation of the a subunit of translation initiation factor 2 (eIF-2a) in mammalian cells leads to inhibition of protein synthesis at the initiation step. Two different mammalian (eIF-2a) kinases have been identified: the doublestranded-RNA-activated inhibitor of translation (DAI) that is activated in response to viral infections and the hemecontrolled repressor (HCR) that is activated in reticulocytes by heme deficiency (for a review, see reference 26). Both kinases phosphorylate eIF-2a on the serine residue at position 51 (Ser-51) (9, 47). The phosphorylation of mammalian eIF-2a inhibits translation initiation by impairing the conversion of eIF-2-GDP to eIF-2-GTP at the completion of each initiation cycle, a reaction carried out by the guanine nucleotide exchange factor eIF-2B. Only the GTP-bound form of eIF-2 is able to form a ternary complex with the initiator tRNAMet and catalyze new rounds of translation initiation (41).GCN2 to increased synthesis of GCN4, a transcriptional activator of numerous genes encoding enzymes involved in amino acid biosynthesis. The kinase activity of GCN2 is required f...

GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.In the yeast Saccharomyces cerevisiae, starvation for any one of several amino acids or a defective aminoacyl-tRNA synthetase leads to increased transcription of over 30 genes encoding amino acid-biosynthetic enzymes in several different pathways (reviewed in reference 19). The coordinate derepression of these biosynthetic pathways is known as general amino acid control. GCN4 is a positive regulatory protein that acts directly to stimulate transcription by binding to 5' noncoding sequences located upstream of each gene subject to the general control.Expression of GCN4 itself is regulated by amino acid availability at the level of translation initiation. This translational control involves four short upstream open reading frames (uORFs) present in the leader of GCN4 mRNA that inhibit translation initiation at the GCN4 start codon under nonstarvation conditions. The inhibitory effect of the four uORFs on GCN4 expression requires trans-acting negative regulators encoded by multiple GCD genes. This group of negative effectors was recently shown to include SUI2 and SUI3, the structural genes for the a and a subunits, respectively, of eucaryotic initiation factor 2 (42). Positive-acting factors encoded by the GCN2 and GCN3 genes are required to stimulate G...

Mutations in three subunits of the SWI/SNF complex and in the Med2p subunit of the SRB/mediator of pol II holoenzyme impaired Gcn4p-activated transcription of HIS3 without reducing Gcn4p-independent transcription of this gene. Recombinant Gcn4p interacted with SWI/SNF and SRB/mediator subunits in cell extracts in a manner dependent on the same hydrophobic clusters in the Gcn4p activation domain; however, higher concentrations of Gcn4p were required for binding to SWI/SNF versus SRB/mediator subunits. In addition, SRB/mediator and SWI/SNF subunits did not coimmunopreciptate from the extracts. These findings, together with the fact that Gcn4p specifically interacted with purified SWI/SNF, strongly suggest that Gcn4p independently recruits SWI/SNF and holoenzyme to its target promoters in the course of activating transcription.

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. The N-terminal 100 amino acids of GCN4 contains a potent activation function that confers high-level transcription in the absence of the centrally located acidic activation domain (CAAD) delineated in previous studies. To identify specific amino acids important for activation by the N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD and screened alleles in vivo for reduced expression of the HIS3 gene. We found four pairs of closely spaced phenylalanines and a leucine residue distributed throughout the N-terminal 100 residues of GCN4 that are required for high-level activation in the absence of the CAAD. Trp, Leu, and Tyr were highly functional substitutions for the Phe residue at position 45. Combined with our previous findings, these results indicate that GCN4 contains seven clusters of aromatic or bulky hydrophobic residues which make important contributions to transcriptional activation at HIS3. None of the seven hydrophobic clusters is essential for activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantial reduction in GCN4 function. Numerous combinations of four or five intact clusters conferred high-level transcription of HIS3. We propose that many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and that the functional contributions of these different segments are cumulative at the HIS3 promoter. On the basis of the primacy of bulky hydrophobic residues throughout the activation domain, we suggest that GCN4 contains multiple sites that mediate hydrophobic contacts with one or more components of the transcription initiation machinery.