scholarly journals Secondary and tertiary structural changes in γδ resolvase: Comparison of the wild-type enzyme, the I11OR mutant, and the C-terminal DNA binding domain in solution

1997 ◽  
Vol 6 (6) ◽  
pp. 1237-1247 ◽  
Author(s):  
Borlan Pan ◽  
Susmita Ghosh ◽  
Gregory P. Mullen ◽  
Zhengwu Deng ◽  
Dingjiang Liu
Keyword(s):  
Dna Binding ◽  
Structural Changes ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Wild Type Enzyme

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors

1993 ◽  
Vol 13 (12) ◽  
pp. 7850-7858
Author(s):  
F Kaspar ◽  
H Klocker ◽  
A Denninger ◽  
A C Cato
Keyword(s):  
Androgen Receptor ◽  
Dna Binding ◽  
Steroid Receptors ◽  
Regulatory Elements ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Binding Studies ◽  
Mutant Receptor ◽  
Alanine Residue

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.

scholarly journals SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations

2020 ◽  
Vol 1864 (1) ◽  
pp. 129440 ◽  
Author(s):  
Ana Sara Gomes ◽  
Helena Ramos ◽  
Sara Gomes ◽  
Joana B. Loureiro ◽  
Joana Soares ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Mutant P53 ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Hotspot Mutations

scholarly journals A single amino acid change in CUP2 alters its mode of DNA binding.

1990 ◽  
Vol 10 (9) ◽  
pp. 4778-4787 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
W Dixon ◽  
T D Tullius ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Dna Binding Domain ◽  
Sequence Motifs ◽  
Wild Type ◽  
Mobility Shift ◽  
Single Amino Acid Change

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

Structure and Dynamics of the Glucocorticoid Receptor DNA-Binding Domain:  Comparison of Wild Type and a Mutant with Altered Specificity†

Biochemistry ◽  
1997 ◽  
Vol 36 (37) ◽  
pp. 11188-11197 ◽  
Author(s):  
Helena Berglund ◽  
Magnus Wolf-Watz ◽  
Thomas Lundbäck ◽  
Susanne van den Berg ◽  
Torleif Härd
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Structure And Dynamics

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing.

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217 ◽  
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Studying the Role of ETV6 in Megakaryopoiesis and Thrombopoiesis Using a Novel CRISPR-Cas9 Halotag Genome Editing Strategy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2468-2468 ◽  
Author(s):  
Marlie H Fisher ◽  
Leila J. Noetzli ◽  
Michael Callaghan ◽  
Walter H. Kahr ◽  
Jesse W Rowley ◽  
...  
Keyword(s):  
Dna Binding ◽  
Research Funding ◽  
Cellular Localization ◽  
Binding Domain ◽  
Clinical Approach ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Transcriptional Targets ◽  
And Function

Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6 lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6 mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6 is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions. We have successfully generated a CRISPR-Cas9 model to edit the genome of ETV6-expressing iPSC derived megakaryocyte cell line (imMKCL) to characterize the role of wild-type ETV6 in megakaryocyte development and elucidate the molecular mechanism driving mutant ETV6 mislocalization, transcriptional dysregulation, and subsequent dysmegakaryopoiesis and thrombocytopenia. In this imMKCL model, we have genetically engineered the cells to express wild-type, P214L, and the DNA binding domain mutations R418G and R369Q ETV6 fused to HALOtag, a reporter protein that can react with ligands carrying a variety of functionalities, including fluorescent labels, affinity handles, and attachment to solid phase, making this novel reporter conducive to immunofluorescence imaging, biochemical pulldown, and ChIPSeq. This system allows us to express wild type and mutant forms of ETV6 in appropriate allele ratios in imMKCL cells and various hematopoietic-relevant cell lines. Using this approach, we detected nuclear localization of wild-type ETV6 and altered cytoplasmic localization of both P214L and R418G ETV6 mutants. We have also demonstrated dimerization between both wild-type and mutant ETV6 in this cell model. Importantly, we have used HALOtag protein immunoprecipitation to demonstrate ETV6 binding to FLI1, another ETS family member and key transcriptional regulator of megakaryocyte development, suggesting that ETV6 and FLI1 cooperate to regulate megakaryopoiesis under homeostatic conditions. Altogether, these data suggest that mutant ETV6 functions as a dominant negative, sequestering wild type ETV6 in the cytoplasm, de-regulating key transcriptional targets for homeostatic megakaryocyte development. Ongoing studies will define the full repertoire of protein interactions and transcriptional targets of wild-type and mutant ETV6. Discoveries from this novel tool will further advance our understanding of normal megakaryocyte and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function to optimize the clinical approach to these patients. Disclosures Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.

scholarly journals Specific Contacts between Residues in the DNA-Binding Domain of the TyrR Protein and Bases in the Operator of thetyrP Gene of Escherichia coli

1999 ◽  
Vol 181 (8) ◽  
pp. 2338-2345 ◽  
Author(s):  
J. S. Hwang ◽  
J. Yang ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Binding Affinities ◽  
Wild Type ◽  
Small Reduction ◽  
Gel Shift Assay ◽  
Gel Shift

ABSTRACT In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051–1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the ±5 positions in the TyrR boxes.

scholarly journals DNA Bending by AraC: a Negative Mutant

1998 ◽  
Vol 180 (16) ◽  
pp. 4227-4232 ◽  
Author(s):  
Beatrice Saviola ◽  
Robert R. Seabold ◽  
Robert F. Schleif
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Dna Bending ◽  
Regulatory Protein ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dna Binding Domain ◽  
Wild Type

ABSTRACT We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the araBAD promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90° whereas the mutant bends DNA by a smaller amount.

scholarly journals Artificial Recruitment of Mediator by the DNA-Binding Domain of Adr1 Overcomes Glucose Repression of ADH2 Expression

2008 ◽  
Vol 28 (8) ◽  
pp. 2509-2516 ◽  
Author(s):  
Elton T. Young ◽  
Christine Tachibana ◽  
Hsin-Wen Ella Chang ◽  
Kenneth M. Dombek ◽  
Erin M. Arms ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Glucose Repression ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Activation Domain ◽  
Unknown Mechanism ◽  
Glucose Depletion ◽  
Nonfermentable Carbon Source

ABSTRACT The transcription factor Adr1 activates numerous genes in nonfermentable carbon source metabolism. An unknown mechanism prevents Adr1 from stably binding to the promoters of these genes in glucose-grown cells. Glucose depletion leads to Snf1-dependent binding. Chromatin immunoprecipitation showed that the Adr1 DNA-binding domain could not be detected at the ADH2 promoter under conditions in which the binding of the full-length protein occurred. This suggested that an activation domain is required for stable binding, and coactivators may stabilize the interaction with the promoter. Artificial recruitment of Mediator tail subunits by fusion to the Adr1 DNA-binding domain overcame both the inhibition of promoter binding and glucose repression of ADH2 expression. In contrast, an Adr1 DNA-binding domain-Tbp fusion did not overcome glucose repression, although it was an efficient activator of ADH2 expression under derepressing conditions. When Mediator was artificially recruited, ADH2 expression was independent of SNF1, SAGA, and Swi/Snf, whereas ADH2 expression was dependent on these factors with wild-type Adr1. These results suggest that in the presence of glucose, the ADH2 promoter is accessible to Adr1 but that other interactions that occur when glucose is depleted do not take place. Artificial recruitment of Mediator appears to overcome this requirement and to allow stable binding and transcription under normally inhibitory conditions.

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