scholarly journals ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences.

1990 ◽  
Vol 10 (1) ◽  
pp. 426-429 ◽  
Author(s):  
C F Evans ◽  
D R Engelke ◽  
D J Thiele
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Fusion Protein ◽  
Binding Sites ◽  
Recognition Sequence ◽  
Multiple Regions

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.

ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences

1990 ◽  
Vol 10 (1) ◽  
pp. 426-429
Author(s):  
C F Evans ◽  
D R Engelke ◽  
D J Thiele
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Fusion Protein ◽  
Binding Sites ◽  
Recognition Sequence ◽  
Multiple Regions

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.

scholarly journals Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (6) ◽  
pp. 2690-2700 ◽  
Author(s):  
M A Huie ◽  
E W Scott ◽  
C M Drazinic ◽  
M C Lopez ◽  
I K Hornstra ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Binding Site ◽  
Gene Function ◽  
Binding Sites ◽  
Sequence Motif ◽  
Wild Type ◽  
Upstream Activating Sequence ◽  
Sequence Elements

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae

1990 ◽  
Vol 10 (7) ◽  
pp. 3797-3800
Author(s):  
B F Ni ◽  
R B Needleman
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Lacz Gene ◽  
Maltose Permease ◽  
Upstream Activating Sequence ◽  
The Subject ◽  
Dna Fragment ◽  
Mal Genes ◽  
Beta Galactosidase

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.

Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae

1988 ◽  
Vol 8 (9) ◽  
pp. 3717-3725
Author(s):  
M Kornuc ◽  
R Altman ◽  
D Harrich ◽  
J Garcia ◽  
J Chao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Binding Domains ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Transactivator Protein

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites

1990 ◽  
Vol 10 (4) ◽  
pp. 1432-1438
Author(s):  
D M Ruden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Schizosaccharomyces Pombe ◽  
Binding Sites ◽  
Transcription Activator ◽  
Integral Number ◽  
Dna Binding Site ◽  
Adh1 Gene

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

scholarly journals Testing for DNA Tracking by MOT1, a SNF2/SWI2 Protein Family Member

1999 ◽  
Vol 19 (1) ◽  
pp. 412-423 ◽  
Author(s):  
David T. Auble ◽  
Susanne M. Steggerda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Family Member ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Tata Binding Protein ◽  
Protein Family ◽  
Dna Complexes ◽  
Dna Templates ◽  
Dependent Interaction

ABSTRACT Proteins in the SNF2/SWI2 family use ATP hydrolysis to catalyze rearrangements in diverse protein-DNA complexes. How ATP hydrolysis is coupled to these rearrangements is unknown, however. One attractive model is that these ATPases are ATP-dependent DNA-tracking enzymes. This idea was tested for the SNF2/SWI2 protein family member MOT1. MOT1 is an essential Saccharomyces cerevisiae transcription factor that uses ATP to dissociate TATA binding protein (TBP) from DNA. By using a series of DNA templates with one or two TATA boxes in combination with binding sites for heterologous DNA binding “roadblock” proteins, the ability of MOT1 to track along DNA was assayed. The results demonstrate that, following ATP-dependent TBP-DNA dissociation, MOT1 dissociates rapidly from the DNA by a mechanism that does not require a DNA end. Template commitment footprinting experiments support the conclusion that ATP-dependent DNA tracking by MOT1 does not occur. These results support a model in which MOT1 drives TBP-DNA dissociation by a mechanism that involves a transient, ATP-dependent interaction with TBP-DNA which does not involve ATP-dependent DNA tracking.

scholarly journals Use of structural DNA properties for the prediction of transcription-factor binding sites in Escherichia coli

2010 ◽  
Vol 39 (2) ◽  
pp. e6-e6 ◽  
Author(s):  
Pieter Meysman ◽  
Thanh Hai Dang ◽  
Kris Laukens ◽  
Riet De Smet ◽  
Yan Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Factor Binding

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae

1992 ◽  
Vol 12 (6) ◽  
pp. 2690-2700
Author(s):  
M A Huie ◽  
E W Scott ◽  
C M Drazinic ◽  
M C Lopez ◽  
I K Hornstra ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Binding Site ◽  
Gene Function ◽  
Binding Sites ◽  
Sequence Motif ◽  
Wild Type ◽  
Upstream Activating Sequence ◽  
Sequence Elements

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

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