Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha 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 GCN2c 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 GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II 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-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 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-2 alpha kinase GCN2.
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PMID:Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases. 144 7

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.
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PMID:Complex formation by positive and negative translational regulators of GCN4. 203 27

The GCN4 protein of S. cerevisiae is a transcriptional activator of amino acid biosynthetic genes which are subject to general amino acid control. GCN3, a positive regulator required for increased GCN4 expression in amino acid-starved cells, is thought to function by antagonism of one or more negative regulators encoded by GCD genes. We isolated gcn3c alleles that lead to constitutively derepressed expression of GCN4 and amino acid biosynthetic genes under its control. These mutations map in the protein-coding sequences and, with only one exception, do not increase the steady-state level of GCN3 protein. All of the gcn3c alleles lead to derepression of genes under the general control in the absence of GCN1 and GCN2, two other positive regulators of GCN4 expression. This finding suggests that GCN3 functions downstream from GCN1 and GCN2 in the general control pathway. In accord with this idea, constitutively derepressing alleles of GCN2 are greatly dependent on GCN3 for their derepressed phenotype. The gcn3c alleles that are least dependent on GCN1 and GCN2 for derepression cause slow-growth under nonstarvation conditions. In addition, all of the gcn3c alleles are less effective than wild-type GCN3 in overcoming the temperature-sensitive lethality associated with certain mutations in the negative regulator GCD2. These results suggest that activation of GCN3 positive regulatory function by the gcn3c mutations involves constitutive antagonism of GCD2 function, leading to reduced growth rates and derepression of GCN4 expression in the absence of amino acid starvation.
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PMID:The translational activator GCN3 functions downstream from GCN1 and GCN2 in the regulatory pathway that couples GCN4 expression to amino acid availability in Saccharomyces cerevisiae. 224 55

The transcriptional activator protein GCN4 is responsible for increased transcription of more than 30 different amino acid biosynthetic genes in response to starvation for a single amino acid. This induction depends on increased expression of GCN4 at the translational level. We show that starvation for purines also stimulates GCN4 translation by the same mechanism that operates in amino acid-starved cells, being dependent on short upstream open reading frames in the GCN4 mRNA leader, the phosphorylation site in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the protein kinase GCN2, and translational activators of GCN4 encoded by GCN1 and GCN3. Biochemical experiments show that eIF-2 alpha is phosphorylated in response to purine starvation and that this reaction is completely dependent on GCN2. As expected, derepression of GCN4 in purine-starved cells leads to a substantial increase in HIS4 expression, one of the targets of GCN4 transcriptional activation. gcn mutants that are defective for derepression of amino acid biosynthetic enzymes also exhibit sensitivity to inhibitors of purine biosynthesis, suggesting that derepression of GCN4 is required for maximal expression of one or more purine biosynthetic genes under 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 purine-starved cells. Our results indicate that the general control response is more global than was previously imagined in terms of the type of nutrient starvation that elicits derepression of GCN4 as well as the range of target genes that depend on GCN4 for transcriptional activation.
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PMID:Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2. 833 37

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.
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PMID:GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2. 849 69

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.
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PMID:Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2. 923 5

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.
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PMID:YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed. 1512