scholarly journals Novel method for identifying sequence-specific DNA-binding proteins.

1985 ◽  
Vol 5 (9) ◽  
pp. 2307-2315 ◽  
Author(s):  
D Levens ◽  
P M Howley
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Dna Sequence ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein S ◽  
Dna Binding Proteins ◽  
Lac Repressor ◽  
Lac Operator ◽  
Beta Galactosidase

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Novel method for identifying sequence-specific DNA-binding proteins

1985 ◽  
Vol 5 (9) ◽  
pp. 2307-2315
Author(s):  
D Levens ◽  
P M Howley
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Dna Sequence ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein S ◽  
Dna Binding Proteins ◽  
Lac Repressor ◽  
Lac Operator ◽  
Beta Galactosidase

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

scholarly journals B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits

1991 ◽  
Vol 11 (2) ◽  
pp. 1156-1160
Author(s):  
C Murre ◽  
A Voronova ◽  
D Baltimore
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Dna Sequence ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Dimerization Domain ◽  
Drosophila Development ◽  
Helix Loop Helix ◽  
Nuclear Factors ◽  
Biochemical Function

Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.

scholarly journals Gel Shift Assay of Nuclear Extracts from Histoplasma capsulatum Demonstrates the Presence of Several DNA Binding Proteins

2002 ◽  
Vol 70 (4) ◽  
pp. 2238-2241 ◽  
Author(s):  
Atanas Ignatov ◽  
Elizabeth J. Keath
Keyword(s):  
Dna Binding ◽  
Fungal Pathogens ◽  
Binding Proteins ◽  
Histoplasma Capsulatum ◽  
Protein S ◽  
Dna Binding Proteins ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Shift Assay ◽  
Gel Shift

ABSTRACT A gel shift assay was optimized to detect several general DNA binding proteins from Histoplasma capsulatum strain G217B. The electrophoretic mobility shift assay (EMSA) technique also detected protein(s) recognizing a pyrimidine-rich motif found in several Histoplasma promoters. Establishment of EMSA conditions provides an important framework to evaluate regulation of homeostatic or phase-specific genes that may influence virulence in Histoplasma and other dimorphic fungal pathogens.

scholarly journals Regulation of Transcription by Hypoxia Requires a Multiprotein Complex That Includes Hypoxia-Inducible Factor 1, an Adjacent Transcription Factor, and p300/CREB Binding Protein

1998 ◽  
Vol 18 (7) ◽  
pp. 4089-4096 ◽  
Author(s):  
Benjamin L. Ebert ◽  
H. Franklin Bunn
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Hypoxia Inducible Factor ◽  
Dna Binding Proteins ◽  
Creb Binding Protein ◽  
Multiprotein Complex ◽  
Hypoxia Inducible Factor 1

ABSTRACT Molecular adaptation to hypoxia depends on the binding of hypoxia-inducible factor 1 (HIF-1) to cognate response elements in oxygen-regulated genes. In addition, adjacent sequences are required for hypoxia-inducible transcription. To investigate the mechanism of interaction between these cis-acting sequences, the multiprotein complex binding to the lactate dehydrogenase A (LDH-A) promoter was characterized. The involvement of HIF-1, CREB-1/ATF-1, and p300/CREB binding protein (CBP) was demonstrated by techniques documenting in vitro binding, in combination with transient transfections that test the in vivo functional importance of each protein. In both the LDH-A promoter and the erythropoietin 3′ enhancer, formation of multiprotein complexes was analyzed by using biotinylated probes encompassing functionally critical cis-acting sequences. Strong binding of p300/CBP required interactions with multiple DNA binding proteins. Thus, the necessity of transcription factor binding sites adjacent to a HIF-1 site for hypoxically inducible transcription may be due to the requirement of p300 to interact with multiple transcription factors for high-affinity binding and activation of transcription. Since it has been found to interact with a wide range of transcription factors, p300 is likely to play a similar role in other genes, mediating interactions between DNA binding proteins, thereby activating stimulus-specific and tissue-specific gene transcription.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

Collation and analyses of DNA-binding protein domain families from sequence and structural databanks

2015 ◽  
Vol 11 (4) ◽  
pp. 1110-1118 ◽  
Author(s):  
Sony Malhotra ◽  
Ramanathan Sowdhamini
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Dna Binding Proteins ◽  
Protein Domain ◽  
Divergent Evolution ◽  
Molecular Function ◽  
Molecular Functions

The distribution of GO molecular functions across different SCOP DNA-binding folds was studied. Majority of the folds were observed to perform more than one molecular function. This supports the notion that majority of DNA-binding proteins might follow divergent evolution.

scholarly journals Fission Yeast Homologs of Human CENP-B Have Redundant Functions Affecting Cell Growth and Chromosome Segregation

2000 ◽  
Vol 20 (8) ◽  
pp. 2852-2864 ◽  
Author(s):  
Mary Baum ◽  
Louise Clarke
Keyword(s):  
Dna Binding ◽  
Fission Yeast ◽  
Dna Sequence ◽  
Chromosome Segregation ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Central Core ◽  
Cell Extracts ◽  
Redundant Functions

ABSTRACT Two functionally important DNA sequence elements in centromeres of the fission yeast Schizosaccharomyces pombe are the centromeric central core and the K-type repeat. Both of these DNA elements show internal functional redundancy that is not correlated with a conserved DNA sequence. Specific, but degenerate, sequences in these elements are bound in vitro by the S. pombeDNA-binding proteins Abp1p (also called Cbp1p) and Cbhp, which are related to the mammalian centromere DNA-binding protein CENP-B. In this study, we determined that Abp1p binds to at least one of its target sequences within S. pombe centromere II central core (cc2) DNA with an affinity (Ks = 7 × 109 M−1) higher than those of other known centromere DNA-binding proteins for their cognate targets. In vivo, epitope-tagged Cbhp associated with centromeric K repeat chromatin, as well as with noncentromeric regions. Likeabp1+/cbp1 +, we found thatcbh + is not essential in fission yeast, but a strain carrying deletions of both genes (Δabp1 Δcbh) is extremely compromised in growth rate and morphology and missegregates chromosomes at very high frequency. The synergism between the two null mutations suggests that these proteins perform redundant functions in S. pombe chromosome segregation. In vitro assays with cell extracts with these proteins depleted allowed the specific assignments of several binding sites for them within cc2 and the K-type repeat. Redundancy observed at the centromere DNA level appears to be reflected at the protein level, as no single member of the CENP-B-related protein family is essential for proper chromosome segregation in fission yeast. The relevance of these findings to mammalian centromeres is discussed.

scholarly journals Isolation and analysis of the yeast TEA1 gene, which encodes a zinc cluster Ty enhancer-binding protein.

1996 ◽  
Vol 16 (1) ◽  
pp. 347-358 ◽  
Author(s):  
W M Gray ◽  
J S Fassler
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Dna Binding Proteins ◽  
Yeast Cells ◽  
Regulatory Sequences ◽  
Zinc Cluster ◽  
Modest Contribution ◽  
C Terminus

A genetic screen for mutants that affect the activity of internal regulatory sequences of Ty retrotransposons led to the identification of a new gene encoding a DNA-binding protein that interacts with the downstream enhancer-like region of Ty1 elements. The TEA1 (Ty enhancer activator) gene sequence predicts a protein of 86.9 kDa whose N terminus contains a zinc cluster and dimerization motif typical of the Gal4-type family of DNA-binding proteins. The C terminus encodes an acidic domain with a net negative charge of -10 and the ability to mediate transcriptional activation. Like other zinc cluster proteins, purified Tea1 was found to bind to a partially palindromic CGGNxCCG repeat motif located in the Ty1 enhancer region. The Ty1 Tea1 binding site has a spacing of 10 and is located near binding sites for the DNA-binding proteins Rap1 and Mcm1. Analysis of the phenotype of tea1 deletion mutants confirmed that the TEA1 gene is required for activation from the internal Ty1 enhancer characterized in this study and makes a modest contribution to normal Ty1 levels in the cell. Hence, Tea1, like Rap1, is a member of a small family of downstream activators in Saccharomyces cerevisiae. Further analysis of the Tea1 protein and its interactions may provide insight into the mechanism of downstream activation in yeast cells.

Sign in / Sign up

Export Citation Format

Share Document