Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase.

I Roussou; G Thireos; B M Hauge

doi:10.1128/mcb.8.5.2132

Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase

Molecular and Cellular Biology ◽

10.1128/mcb.8.5.2132-2139.1988 ◽

1988 ◽

Vol 8 (5) ◽

pp. 2132-2139

Author(s):

I Roussou ◽

G Thireos ◽

B M Hauge

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Protein Kinase ◽

Dna Sequences ◽

Transcriptional Activation ◽

Translational Regulation ◽

Catalytic Domain ◽

Amino Acid Starvation ◽

Protein Kinase Activity ◽

Regulatory Circuit

GCN4 protein mediates the transcriptional activation of amino acid biosynthetic genes in Saccharomyces cerevisiae by specifically binding to DNA sequences in their 5'-regulatory regions. GCN4 expression is regulated at the level of translation, with translational derepression occurring under conditions of amino acid starvation. The product of the GCN2 gene is essential for translational derepression of GCN4. Sequence analysis of the GCN2 gene reveals that the GCN2 protein has a domain highly homologous to the catalytic domain of all known protein kinases. Furthermore, gcn2 strains are deficient in a protein kinase activity corresponding to a protein with the calculated molecular weight deduced from the GCN2 open reading frame. Therefore it is likely that GCN2 encodes a protein kinase, which may be directly involved in translational regulation of the GCN4 mRNA. Transcription of the GCN2 gene is increased when cells are cultured in amino acid starvation medium. This transcriptional activation is mediated by the GCN4 protein, which binds to the promoter region of the GCN2 gene. Thus, this system is modulated by a transcriptional-translational regulatory circuit, which is activated by amino acid starvation. Activation is not the result of a simple quantitative increase of either one of the identified components of the circuit.

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Translational Control of Protein Kinase Cη by Two Upstream Open Reading Frames

Molecular and Cellular Biology ◽

10.1128/mcb.01044-09 ◽

2009 ◽

Vol 29 (22) ◽

pp. 6140-6148 ◽

Cited By ~ 15

Author(s):

Hadas Raveh-Amit ◽

Adva Maissel ◽

Jonathan Poller ◽

Liraz Marom ◽

Orna Elroy-Stein ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Translational Control ◽

Translational Regulation ◽

Normal Growth ◽

Amino Acid Starvation ◽

Open Reading Frames ◽

Pkc Isoforms ◽

Upstream Open Reading Frames ◽

Reading Frames

ABSTRACT Protein kinase C (PKC) represents a family of serine/threonine kinases that play a central role in the regulation of cell growth, differentiation, and transformation. Posttranslational control of the PKC isoforms and their activation have been extensively studied; however, not much is known about their translational regulation. Here we report that the expression of one of the PKC isoforms, PKCη, is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation, the latter causing a marked increase in its protein levels. The 5′ untranslated region (5′ UTR) of PKCη is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5′ UTR and show their effect in suppressing the expression of PKCη in MCF-7 growing cells. While the two uORFs function as repressive elements that maintain low basal levels of PKCη in growing cells, they are required for its enhanced expression upon amino acid starvation. We show that the translational regulation during stress involves leaky scanning and is dependent on eIF-2α phosphorylation by GCN2. Our work further suggests that translational regulation could provide an additional level for controlling the expression of PKC family members, being more common than currently recognized.

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Nucleosome Position-Dependent and -Independent Activation of HIS7 Expression in Saccharomyces cerevisiae by Different Transcriptional Activators

Eukaryotic Cell ◽

10.1128/ec.2.5.876-885.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 876-885 ◽

Cited By ~ 4

Author(s):

Oliver Valerius ◽

Cornelia Brendel ◽

Claudia Wagner ◽

Sven Krappmann ◽

Fritz Thoma ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factors ◽

Amino Acid ◽

Transcriptional Activation ◽

Intergenic Region ◽

Nucleosome Position ◽

Amino Acid Starvation ◽

Transcriptional Activators ◽

Promoter Regions ◽

Independent Mechanism

ABSTRACT ARO4 and HIS7 are two tandemly orientated genes of Saccharomyces cerevisiae that are transcribed into the same direction. The ARO4 terminator and the HIS7 promoter regions are sensitive to Micrococcus nuclease (Mnase) and separated by a positioned nucleosome. The HIS7 promoter is target for the transcription factors Gcn4p and Bas1p/Bas2p that activate its transcription upon amino acid starvation and purine limitation, respectively. Activation of the HIS7 gene by Gcn4p overexpression but not by Bas1p/Bas2p releases an ordered nucleosome distribution to yield increased Mnase sensitivity throughout the intergenic region. This remodeling is SNF2 dependent but mostly GCN5 independent. Accordingly, SNF2 is necessary for the Gcn4p-mediated transcriptional activation of the HIS7 gene. GCN5 is required for activation upon adenine limitation by Bas1p/Bas2p. Our data suggest that activation of HIS7 transcription by Gcn4p and Bas1p/Bas2p is supported by a nucleosome position-dependent and -independent mechanism, respectively. Whereas Gcn4p activation causes Swi/Snf-mediated remodeling of the nucleosomal architecture at the HIS7 promoter, the Bas1p/Bas2p complex presumably activates in combination with Gcn5p-dependent histone acetylation.

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Phosphorylation by Cak1 Regulates the C-Terminal Domain Kinase Ctk1 in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.25.10.3906-3913.2005 ◽

2005 ◽

Vol 25 (10) ◽

pp. 3906-3913 ◽

Cited By ~ 25

Author(s):

Denis Ostapenko ◽

Mark J. Solomon

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Stationary Phase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Kinase Activity ◽

Protein Kinase Activity ◽

Transcriptional Elongation ◽

Terminal Domain

ABSTRACT Ctk1 is a Saccharomyces cerevisiae cyclin-dependent protein kinase (CDK) that assembles with Ctk2 and Ctk3 to form an active protein kinase complex, CTDK-I. CTDK-I phosphorylates Ser2 within the RNA polymerase II C-terminal domain, an activity that is required for efficient transcriptional elongation and 3′ RNA processing. Ctk1 contains a conserved T loop, which undergoes activating phosphorylation in other CDKs. We show that Ctk1 is phosphorylated on Thr-338 within the T loop. Mutation of this residue abolished Ctk1 kinase activity in vitro and resulted in a cold-sensitive phenotype. As with other yeast CDKs undergoing T-loop phosphorylation, Ctk1 phosphorylation on Thr-338 was dependent on the Cak1 protein kinase. Ctk1 isolated from cak1Δ cells was unphosphorylated and exhibited low protein kinase activity. Moreover, Cak1 directly phosphorylated Ctk1 in vitro. Unlike wild-type cells, cells expressing Ctk1T338A delayed growth at early stationary phase, did not show the increase in Ser2 phosphorylation that normally accompanies the transition from rapid growth to stationary phase, and had compromised transcriptional activation of two stationary-phase genes, CTT1 and SPI1. Therefore, Ctk1 phosphorylation on Thr-338 is carried out by Cak1 and is required for normal gene transcription during the transition into stationary phase.

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Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2653-2663.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2653-2663 ◽

Cited By ~ 1

Author(s):

J F Cannon ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Cyclic Amp ◽

Dna Sequences ◽

Regulatory Subunit ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Ras Genes ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS1 or RAS2) in Saccharomyces cerevisiae. We cloned SRA1 and SRA3 and determined their DNA sequences. SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent protein kinase and therefore is identical to REG1 and BCY1. This gene is not essential, but its deletion confers many traits: reduction of glycogen accumulation, temperature sensitivity, reduced growth rate on maltose and sucrose, inability to grow on galactose and nonfermentable carbon sources, and nitrogen starvation intolerance. SRA3 is homologous to protein kinases that phosphorylate serine and threonine and likely encodes the catalytic subunit of the cAMP-dependent protein kinase. The wild-type SRA3 gene either triplicated in the chromosome or on episomal, low-copy plasmids behaves like spontaneous dominant SRA3 mutations by suppressing ras2-530 (RAS2::LEU2 disruption), cdc25, and cdc35 mutations. These findings indicate that the yeast RAS genes are dispensable if there is constitutive cAMP-dependent protein kinase activity.

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Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2653 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2653-2663 ◽

Cited By ~ 154

Author(s):

J F Cannon ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Cyclic Amp ◽

Dna Sequences ◽

Regulatory Subunit ◽

Protein Kinase Activity ◽

Dependent Protein Kinase ◽

Ras Genes ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

Mutations in the SRA1 or SRA3 gene eliminate the requirement for either RAS gene (RAS1 or RAS2) in Saccharomyces cerevisiae. We cloned SRA1 and SRA3 and determined their DNA sequences. SRA1 encodes the regulatory subunit of the cyclic AMP (cAMP)-dependent protein kinase and therefore is identical to REG1 and BCY1. This gene is not essential, but its deletion confers many traits: reduction of glycogen accumulation, temperature sensitivity, reduced growth rate on maltose and sucrose, inability to grow on galactose and nonfermentable carbon sources, and nitrogen starvation intolerance. SRA3 is homologous to protein kinases that phosphorylate serine and threonine and likely encodes the catalytic subunit of the cAMP-dependent protein kinase. The wild-type SRA3 gene either triplicated in the chromosome or on episomal, low-copy plasmids behaves like spontaneous dominant SRA3 mutations by suppressing ras2-530 (RAS2::LEU2 disruption), cdc25, and cdc35 mutations. These findings indicate that the yeast RAS genes are dispensable if there is constitutive cAMP-dependent protein kinase activity.

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Protein kinase A mediates growth-regulated expression of yeast ribosomal protein genes by modulating RAP1 transcriptional activity

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1920-1928.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1920-1928

Author(s):

C Klein ◽

K Struhl

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Carbon Source ◽

Ribosomal Protein ◽

Binding Sites ◽

Transcriptional Activity ◽

Amino Acid Starvation ◽

Ribosomal Protein Genes ◽

Regulated Expression ◽

Kinase A

Yeast ribosomal protein genes are coordinately regulated as a function of cell growth; RNA levels decrease during amino acid starvation but increase following a carbon source upshift. Binding sites for RAP1, a multifunctional transcription factor, are present in nearly all ribosomal protein genes and are associated with growth rate regulation. We show that ribosomal protein mRNA levels are increased twofold in strains that have constitutively high levels of cyclic AMP-dependent protein kinase (protein kinase A [PKA]) activity. The PKA-dependent induction requires RAP1 binding sites, and it reflects increased transcriptional activation by RAP1. Growth-regulated transcription of ribosomal protein genes strongly depends on the ability to regulate PKA activity. Cells with constitutively high PKA levels do not show the transcriptional decrease in response to amino acid starvation. Conversely, in cells with constitutively low PKA activity, ribosomal protein mRNAs levels are lower and largely uninducible upon carbon source upshift. We suggest that modulation of RAP1 transcriptional activity by PKA accounts for growth-regulated expression of ribosomal protein genes.

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Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.5034-5044.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 5034-5044

Author(s):

J L Celenza ◽

M Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Mutational Analysis ◽

Gene Dosage ◽

Regulatory System ◽

Kinase Domain ◽

Regulatory Function ◽

Protein Kinase Activity ◽

Glucose Limitation

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

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Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.5099-5111.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 5099-5111

Author(s):

R J Rolfes ◽

A G Hinnebusch

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Transcriptional Activation ◽

Phosphorylation Site ◽

Transcriptional Activator ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Single Amino Acid ◽

Biosynthetic Genes ◽

Transcriptional Activator Protein

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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DNA SEQUENCES OF FRAMESHIFT AND OTHER MUTATIONS INDUCED BY ICR-170 IN YEAST

Genetics ◽

10.1093/genetics/111.2.233 ◽

1985 ◽

Vol 111 (2) ◽

pp. 233-241

Author(s):

Joachim F Ernst ◽

D Michael Hampsey ◽

Fred Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Sequence Analysis ◽

Genetic Analysis ◽

Dna Sequences ◽

Induced Mutations ◽

Sequence Analyses ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Frameshift Mutations

ABSTRACT ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G·C additions at sites containing monotonous runs of three G·C base pairs. However, some (see PDF) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G·C base pairs.

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