Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation

The product of the LYS14 gene of Saccharomyces cerevisiae activates the transcription of at least four genes involved in lysine biosynthesis. Physiological and genetic studies indicate that this activation is dependent on the inducer alpha-aminoadipate semialdehyde, an intermediate of the pathway. The gene LYS14 was sequenced and, from its nucleotide sequence, predicted to encode a 790-amino-acid protein carrying a cysteine-rich DNA-binding motif of the Zn(II)2Cys6 type in its N-terminal portion. Deletion of this N-terminal portion including the cysteine-rich domain resulted in the loss of LYS14 function. To test the function of Lys14 as a transcriptional activator, this protein without its DNA-binding motif was fused to the DNA-binding domain of the Escherichia coli LexA protein. The resulting LexA-Lys14 hybrid protein was capable of activating transcription from a promoter containing a lexA operator, thus confirming the transcriptional activation function of Lys14. Furthermore, evidence that this function, which is dependent on the presence of alpha-aminoadipate semialdehyde, is antagonized by lysine was obtained. Such findings suggest that activation by alpha-aminoadipate semialdehyde and the apparent repression by lysine are related mechanisms. Lysine possibly acts by limiting the supply of the coinducer, alpha-aminoadipate semialdehyde.

Download Full-text

Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2576-2582.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2576-2582

Author(s):

A B Clark ◽

C C Dykstra ◽

A Sugino

Keyword(s):

Saccharomyces Cerevisiae ◽

Specific Activity ◽

Intragenic Recombination ◽

Strand Transfer ◽

Homologous Pairing ◽

Spore Viability ◽

Transfer Protein ◽

Acid Protein ◽

Dna Strand ◽

Amino Acid Protein

DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.

Download Full-text

Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2502 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2502-2510 ◽

Cited By ~ 83

Author(s):

F Randez-Gil ◽

N Bojunga ◽

M Proft ◽

K D Entian

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Transcriptional Activation ◽

Phosphoenolpyruvate Carboxykinase ◽

Glucose Repression ◽

Sodium Dodecyl ◽

Binding Motif ◽

Gluconeogenic Enzymes ◽

Zinc Cluster ◽

Activating Function

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

Download Full-text

Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.123-132.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 123-132

Author(s):

A D Sharrocks ◽

H Gille ◽

P E Shaw

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Serum Response Factor ◽

Alpha Helix ◽

Site Directed Mutagenesis ◽

Binding Motif ◽

Amino Acid Peptide ◽

Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

Download Full-text

The Activity of Mammalian brm/SNF2α Is Dependent on a High-Mobility-Group Protein I/Y-Like DNA Binding Domain

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.3931 ◽

1999 ◽

Vol 19 (6) ◽

pp. 3931-3939 ◽

Cited By ~ 56

Author(s):

Brigitte Bourachot ◽

Moshe Yaniv ◽

Christian Muchardt

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

High Mobility ◽

High Mobility Group ◽

Binding Motif ◽

High Mobility Group Protein ◽

Interaction Domain ◽

Dna Binding Domains ◽

Binding Domains ◽

Group Protein

ABSTRACT The mammalian SWI-SNF complex is a chromatin-remodelling machinery involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2α and BRG-1/SNF2β. These two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105Rb repression of E2F transcriptional activity. In the present study, we have examined the ability of various brm/SNF2α deletion mutants to reverse the transformed phenotype ofras-transformed fibroblasts. Deletions within the p105Rb LXCXE binding motif or the conserved bromodomain had only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105Rb interaction domain, appeared to be essential in this assay. This region was also required for cooperation of brm/SNF2α with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2α to chromatin.

Download Full-text

The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.611 ◽

1991 ◽

Vol 11 (2) ◽

pp. 611-619 ◽

Cited By ~ 50

Author(s):

J T Olesen ◽

J D Fikes ◽

L Guarente

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Transcriptional Activation ◽

Core Protein ◽

Protein Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Acid Protein ◽

Ccaat Box

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

Download Full-text

Yeast Coactivator MBF1 Mediates GCN4-Dependent Transcriptional Activation

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.4971 ◽

1998 ◽

Vol 18 (9) ◽

pp. 4971-4976 ◽

Cited By ~ 70

Author(s):

Ken-ichi Takemaru ◽

Satoshi Harashima ◽

Hitoshi Ueda ◽

Susumu Hirose

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Nuclear Receptor ◽

Transcriptional Activation ◽

Crucial Role ◽

Tata Binding Protein ◽

Binding Region

ABSTRACT Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery. The coactivator multiprotein bridging factor 1 (MBF1) was originally identified as a bridging molecule that connects theDrosophila nuclear receptor FTZ-F1 and TATA-binding protein (TBP). The MBF1 sequence is highly conserved across species fromSaccharomyces cerevisiae to human. Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP. These findings indicate that the coactivator MBF1 functions by recruiting TBP to promoters where DNA-binding regulators are bound.

Download Full-text

nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5735 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5735-5745 ◽

Cited By ~ 55

Author(s):

G F Yuan ◽

Y H Fu ◽

G A Marzluf

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Neurospora Crassa ◽

Transcriptional Activation ◽

Regulatory Gene ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Dna Binding Domain ◽

Amino Terminal ◽

Rich Domain

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

Download Full-text

The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.611-619.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 611-619

Author(s):

J T Olesen ◽

J D Fikes ◽

L Guarente

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Transcriptional Activation ◽

Core Protein ◽

Protein Domain ◽

Yeast Saccharomyces Cerevisiae ◽

Acid Protein ◽

Ccaat Box

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

Download Full-text

A Novel Rb- and p300-Binding Protein Inhibits Transactivation by MyoD

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8903-8915.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8903-8915 ◽

Cited By ~ 83

Author(s):

W. Robb MacLellan ◽

G. Xiao ◽

M. Abdellatif ◽

Michael D. Schneider

Keyword(s):

Cell Cycle ◽

Skeletal Muscle ◽

Binding Motif ◽

Tissue Specific ◽

Yeast Two Hybrid System ◽

C Terminus ◽

Acid Protein ◽

P300 Binding ◽

Amino Acid Protein ◽

Novel Protein

ABSTRACT The retinoblastoma protein (Rb) regulates both the cell cycle and tissue-specific transcription, by modulating the activity of factors that associate with its A-B and C pockets. In skeletal muscle, Rb has been reported to regulate irreversible cell cycle exit and muscle-specific transcription. To identify factors interacting with Rb in muscle cells, we utilized the yeast two-hybrid system, using the A-B and C pockets of Rb as bait. A novel protein we have designated E1A-like inhibitor of differentiation 1 (EID-1), was the predominant Rb-binding clone isolated. It is preferentially expressed in adult cardiac and skeletal muscle and encodes a 187-amino-acid protein, with a classic Rb-binding motif (LXCXE) in its C terminus. Overexpression of EID-1 in skeletal muscle inhibited tissue-specific transcription. Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD independent of G1 exit and, surprisingly, was potentiated by a mutation that prevents EID-1 binding to Rb. Inhibition of MyoD may be explained by EID-1's ability to bind and inhibit p300's histone acetylase activity, an essential MyoD coactivator. Thus, EID-1 binds both Rb and p300 and is a novel repressor of MyoD function.

Download Full-text