Identification of a Low Specificity, Oxygen, Heme, and Growth Phase-Regulated DNA Binding Activity in Saccharomyces cerevisiae

1995 ◽  
Vol 216 (2) ◽  
pp. 458-466 ◽  
Author(s):  
R.M. Wright ◽  
S.L. Simpson ◽  
B.D. Lanoil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Growth Phase ◽  
Binding Activity ◽  
Dna Binding Activity

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites

1990 ◽  
Vol 10 (2) ◽  
pp. 859-862
Author(s):  
G M Santangelo ◽  
J Tornow
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Heterologous Gene ◽  
Dna Binding Activity ◽  
Glycolytic Gene

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

scholarly journals Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

1990 ◽  
Vol 10 (2) ◽  
pp. 859-862 ◽  
Author(s):  
G M Santangelo ◽  
J Tornow
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Heterologous Gene ◽  
Dna Binding Activity ◽  
Glycolytic Gene

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

scholarly journals Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913 ◽  
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

scholarly journals LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes.

1987 ◽  
Vol 7 (8) ◽  
pp. 2708-2717 ◽  
Author(s):  
P Friden ◽  
P Schimmel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Coding Region ◽  
General Amino Acid Control ◽  
Dna Binding Activity ◽  
Cysteine Motif ◽  
Cell Extracts

Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine. We report here an analysis of LEU3, a putative leucine-specific regulatory locus. We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3. We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids. A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region. The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation. We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2. Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1.

LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes

1987 ◽  
Vol 7 (8) ◽  
pp. 2708-2717
Author(s):  
P Friden ◽  
P Schimmel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Coding Region ◽  
General Amino Acid Control ◽  
Dna Binding Activity ◽  
Cysteine Motif ◽  
Cell Extracts

Although the majority of genes for amino acid biosynthesis which have been examined are under general amino acid control, LEU1 and LEU2 of Saccharomyces cerevisiae respond specifically to leucine. We report here an analysis of LEU3, a putative leucine-specific regulatory locus. We show that LEU3 is necessary for expression of wild-type levels of LEU1- and LEU2-specific RNAs and, further, that the levels of LEU4-specific transcripts are also affected by LEU3. We cloned LEU3 and showed by DNA sequence analysis that it contained an open reading frame of 886 amino acids. A striking feature of the predicted LEU3 protein was a cluster of acidic amino acids (19 of 20) located in the C-terminal half of the coding region. The protein also had a repeated cysteine motif which was conserved in a number of other yeast proteins implicated in gene regulation. We show that whole-cell extracts contained a LEU3-dependent DNA-binding activity that interacted with the 5' region of LEU2. Subdivision of the LEU2 5' region established that the LEU3-dependent DNA-binding activity interacted with the segment which had the previously reported homology with LEU1.

scholarly journals A novel allele of HAP1 causes uninducible expression of HEM13 in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 819-831 ◽  
Author(s):  
S C Ushinsky ◽  
T Keng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Mutant Allele ◽  
Binding Activity ◽  
The Other ◽  
Wild Type ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Allele Expression ◽  
Novel Allele

Abstract Transcription of HEM13 in Saccharomyces cerevisiae is repressed by heme and oxygen. We have isolated two mutants in which expression of HEM13 is aberrant. The mutant alleles in these strains represent two different alleles of HAP1. HAP1 encodes an activator protein whose DNA binding activity is stimulated by heme, and is required for the transcription of CYC1, ROX1 and a number of other heme-dependent genes. One of our mutant alleles confers a phenotype much like that of the hap1::LEU2 allele. Expression of HEM13 in a strain with this mutation is elevated under repressing conditions and not fully inducible in the absence of heme. The other mutant allele of HAP1 we uncovered confers a novel phenotype. A strain containing this allele exhibits heme-independent expression of CYC1 and ROX1 and uninducible expression of HEM13 and ANB1. The mutation associated with this novel allele of HAP1 was localized to a glycine to aspartate change in amino acid 235 of HAP1, between the DNA binding and heme responsive domains. DNA binding assays demonstrated that the protein made from this HAP1 allele retains the ability to bind DNA, but that unlike wild-type HAP1 protein, this binding is not stimulated by heme.

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