Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0038187 (starvation)
24,951 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.
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PMID:Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae. 353 30

The GCN4 gene encodes a positive effector of amino acid biosynthetic genes in Saccharomyces cerevisiae. Genetic analysis has suggested that GCN4 is regulated by a hierarchy of interacting positive and negative effectors in response to amino acid starvation. Results presented here for a GCN4-lacZ gene fusion support this regulatory model and suggest that the regulators of GCN4 exert their effects primarily at the level of translation of GCN4 mRNA. Both the GCN2 and GCN3 products appear to stimulate translation of GCN4 mRNA in response to amino acid starvation, because a recessive mutation in either gene blocked derepression of GCN4-lacZ fusion enzyme levels but did not reduce the fusion transcript level relative to that in wild-type cells grown in the same conditions. The GCD1 product appears to inhibit translation of GCN4 mRNA because under certain growth conditions, the gcd1-101 mutation led to derepression of the GCN4-lacZ fusion enzyme level in the absence of any increase in the fusion transcript level. In addition, the gcd1-101 mutation suppressed the low translational efficiency of GCN4-lacZ mRNA observed in gcn2- and gcn3- cells. A deletion of four small open reading frames in the 5' leader of GCN4-lacZ mRNA mimicked the effect of a gcd1 mutation and derepressed translation of the fusion transcript in the absence of either starvation conditions or the GCN2 and GCN3 products. By contrast, in a gcd1- strain, the deletion resulted in little additional increase in the translational efficiency of the fusion transcript. These results suggest that GCD1 mediates the translational repression normally exerted by the GCN4 leader sequences and that GCN2 and GCN3 antagonize these negative elements in response to amino acid starvation. The effects of the trans-acting mutations on the translation of GCN4-lacZ mRNA remained intact even when transcription of the fusion gene was placed under the control of the S. cerevisiae GAL1 transcriptional control element.
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PMID:A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. 391 40

The GCN4 gene encodes a positive regulator of unlinked amino acid biosynthetic genes in yeast. I present evidence that the GCN4 gene is itself regulated by amino acid availability and that the regulation occurs at the translational level. A GCN4-lacZ fusion was used as a measure of the expression of GCN4 gene product. Starvation for histidine leads to derepression of the fusion enzyme in the wild type but not in a gcn2- strain. The gcn2- mutation does not reduce fusion transcript levels relative to wild type, suggesting that the product of GCN2 functions as an activator of GCN4 translation. The GCN4 transcript has a 5' leader that is approximately equal to 600 nucleotides long and contains four small open reading frames. A deletion of the small open reading frames results in constitutive derepression of fusion enzyme levels as the result of an approximately equal to 10-fold increase in the efficiency of translation of the fusion transcript. The deletion suppresses the requirement for GCN2 function. These results suggest that the GCN4 5' leader acts in cis to repress GCN4 translation and that GCN4 translation increases in response to amino acid starvation as the result of GCN2 antagonism of the repressing sequences in the GCN4 5' leader.
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PMID:Evidence for translational regulation of the activator of general amino acid control in yeast. 638 4

Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.
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PMID:The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. 762 40

Phosphorylation of translation initiation factor 2 alpha is a highly conserved mechanism for down-regulating protein synthesis in response to starvation or stress. The yeast eIF-2 alpha kinase GCN2 is stimulated by deprivation for amino acids or purines. In addition to inhibiting general protein synthesis, GCN2 specifically stimulates translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. HRI is an eIF-2 alpha kinase that is activated in rabbit reticulocytes by heme-deprivation and stress conditions that elicit the heat-shock response. The eIF-2 alpha kinase DAI is activated by double-stranded RNA during viral infections and is an important component of the interferon response. DAI has also been implicated as a tumor suppressor. These protein kinases provide an important means of coupling the rate of protein synthesis and cell division to environmental conditions.
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PMID:The eIF-2 alpha kinases: regulators of protein synthesis in starvation and stress. 771 Dec 90

Regulation of the effective activity of eukaryotic initiation factor 2 (eIF-2) in protein synthesis is known to involve phosphorylation of its alpha subunit. Two mammalian enzymes, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI), phosphorylate Ser-51 of the alpha subunit, thereby inhibiting the exchange of bound nucleotides on, and thus the recycling of, eIF-2. In Saccharomyces cerevisiae, the equivalent serine seems to be phosphorylated by the GCN2 protein kinase, which is activated by amino acid starvation. However, in the present paper we show that this is not the only site of phosphorylation in yeast eIF-2 alpha. We report the preparation of recombinant yeast eIF-2 alpha from Escherichia coli and its use in in vitro phosphorylation studies. Mammalian HCR and dsI are shown to phosphorylate specifically Ser-51 of yeast eIF-2 alpha, whereas extracts from yeast cells do not. Instead, at least one of three serine residue in the acidic C-terminal region of this protein is phosphorylated by fractions of yeast possessing casein kinase activities 1 and 2. A triple Ser-->Ala mutant form of yeast eIF-2 alpha was found to be no longer phosphorylated by either of the yeast (or mammalian) casein kinase activities in vitro. Isoelectric focusing of yeast extracts confirmed that the mutated sites normally act as sites of phosphorylation in vivo. The same mutant was used to show that the three sites have no essential function under normal physiological conditions in yeast. In contrast, deletion of the 13 amino acid long C-terminal region of eIF-2 alpha, including the three phosphorylation sites, led to derepression of GCN4 in vivo. Thus removal of the short, highly acidic C-terminal region of eIF-2 alpha has the same regulatory effect on translational (re)initiation as phosphorylation of the Ser-51 residue of the wild-type protein. This result provides new insight into the role of eIF-2 alpha activity in the regulation of translational (re-) initiation.
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PMID:The highly acidic C-terminal region of the yeast initiation factor subunit 2 alpha (eIF-2 alpha) contains casein kinase phosphorylation sites and is essential for maintaining normal regulation of GCN4. 774 63

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of eIF-2 alpha. Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORF1 fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of eIF-2 that delivers charged initiator tRNA(iMet) to the ribosome. When the levels of eIF-2.GTP.Met-tRNA(iMet) ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of eIF-2 by the protein kinase GCN2 decreases the concentration of eIF-2.GTP.Met-tRNA(iMet) complexes by inhibiting the guanine nucleotide exchange factor for eIF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of eIF-2.
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PMID:Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2. 793 12

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.
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PMID:Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation. 816 76

The GCN2 (general control kinase 2) protein is an eIF2-alpha (eukaryotic initiation factor alpha) kinase which mediates translational derepression of the yeast general control transcriptional activator, GCN4, upon amino-acid starvation. We isolated and characterized GCN2 mutations differentially affecting GCN2 function. Mutations mapping in, or close to, the ATP-binding site of the kinase moiety result in constitutively activated GCN2 molecules. A C-terminal regulatory mutation dramatically affects translation initiation rates resulting in pleiotropic phenotypes. The effect of mutations in both regions were found to depend on eIF2-alpha phosphorylation. We have demonstrated that GCN2 mutants have altered autophosphorylation activities in vitro, depending on the presence or absence of a wild-type GCN2 gene and that GCN2 elutes in gel-filtration chromatography fractions with high apparent molecular mass. Both these genetic and biochemical findings suggest that GCN2 functioning might involve polymerization to form dimers or tetramers.
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PMID:Genetic and biochemical evidence for yeast GCN2 protein kinase polymerization. 820 May 34

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


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