scholarly journals A Single Amino Acid Change in the First Zinc Finger of the DNA Binding Domain of the Glucocorticoid Receptor Regulates Differential Promoter Selectivity

2004 ◽  
Vol 279 (38) ◽  
pp. 39279-39288 ◽  
Author(s):  
Brian M. Necela ◽  
John A. Cidlowski
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Zinc Finger ◽  
Amino Acid Change ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Dna Binding Domain ◽  
Single Amino Acid Change

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.

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.

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis

1988 ◽  
Vol 8 (9) ◽  
pp. 3726-3733
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Carboxyl Terminal

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

Pre-Donor Evaluation of an HLA Matched Sibling Identifies a Novel Inherited RUNX1 Mutation Encoding a Missense Mutation Found Outside of the RUNT Domain in Familial Platelet Disorder

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2709-2709 ◽  
Author(s):  
Jacqueline S Garcia ◽  
Jozef Madzo ◽  
Devin Cooper ◽  
Sarah A Jackson ◽  
Kenan Onel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Amino Acid Change ◽  
Platelet Disorder ◽  
Familial Platelet Disorder ◽  
Runx1 Mutation

Abstract Abstract 2709 Introduction: RUNX1 is a critical transcription factor in the regulation of normal hematopoiesis. Inherited RUNX1 mutations have been identified as the culprit genetic lesion in Familial Platelet Disorder (FPD; OMIM 601399), a rare autosomal dominant condition with a propensity to myeloid malignancy. The spectrum of RUNX1 mutations causing the FPD/acute myeloid leukemia (AML) syndrome includes frameshift and termination mutations detected throughout the gene, and missense mutations clustered within the highly conserved RUNT homology domain (RHD), which is responsible for both DNA binding and heterodimerization with CBFβ/PEBP2β, the non-DNA binding regulatory subunit. We present a new FPD/AML pedigree with a novel missense mutation leading to a single amino acid change, L56S. This L56S mutation is the first reported point mutation in this syndrome to be found outside of the RHD. Patients and Methods: Our new pedigree involves a 41-year-old man (proband) diagnosed with myelodysplastic syndrome (MDS, specifically refractory anemia with excess blasts type-2) with a normal karyotype. He was initiated on azacitidine, which was administered on a seven-day treatment schedule every four weeks. Bone marrow biopsy analysis after six monthly cycles of azacitidine showed persistent MDS, with similar findings after a total of ten monthly cycles. Given his lack of a clinical response, his young age and good performance status, he was referred to The University of Chicago for allogeneic hematopoietic stem cell transplantation (HCT). Routine pre-transplant evaluation revealed mild thrombocytopenia (platelets = 123,000 K/μl) in his HLA-matched brother. In addition, his father was reported to have thrombocytopenia. Clinical concern for an inherited condition initiated the investigation for a RUNX1 mutation in the family. Results: We sequenced full-length cDNA synthesized from leukocyte-derived RNA collected from the proband's sibling with thrombocytopenia, and detected a novel missense germline mutation in exon 4 at nucleotide position 371, causing a T to C mutation leading to a single amino acid change in the RUNX1 protein, L56S. This amino acid substitution is located N-terminal to the RHD (aa 76–209). RUNX1 sequencing of the proband with MDS demonstrated the same mutation. The RUNX1 RHD and the transactivation domain remain intact in this mutant. Initial transactivation assays using a luciferase reporter assay performed in triplicate demonstrated similar levels of activation as wild-type RUNX1. Corresponding Western blot analysis showed similar levels of protein expression of both wild-type RUNX1 and mutant RUNX1 transfected cell lines using an anti-RUNX1-antibody. Current studies include determination of the transactivation ability of mutant RUNX1 with its heterodimerization partner, CBFβ/PEBP2β, testing the DNA binding ability of this RUNX1 mutant by electrophoretic mobility shift assay, and analysis of the RUNX1 cDNA for an acquired biallelic mutation in leukocytes collected from the proband's bone marrow aspirate at the time of diagnosis of bone marrow malignancy. Conclusions: FPD/AML is likely an underreported condition. Clinical suspicion for this inherited syndrome may be raised by the presence of mild to moderate thrombocytopenia in healthy siblings, and should lead to prompt screening for germline RUNX1 mutations to confirm an inherited predisposition and to prevent siblings carrying RUNX1 mutations from being selected as HCT donors. In vitro studies of identified RUNX1 mutations may elucidate potential mechanisms involved in the pathogenesis of the FPD/AML syndrome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Insertion of an Amino Acid in the DNA-Binding Domain of the Glucocorticoid Receptor as a Result of Alternative Splicing

1999 ◽  
Vol 84 (11) ◽  
pp. 4283-4286 ◽  
Author(s):  
Caroline Rivers ◽  
Andrew Levy ◽  
Jerry Hancock ◽  
Stafford Lightman ◽  
Michael Norman
Keyword(s):  
Alternative Splicing ◽  
Amino Acid ◽  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain

The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

scholarly journals Biochemical characterization of human Ecdysoneless reveals a role in transcriptional regulation

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Jun Hyun Kim ◽  
Channabasavaiah Basavaraju Gurumurthy ◽  
Hamid Band ◽  
Vimla Band
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Biochemical Characterization ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Luciferase Reporter ◽  
Dna Binding Domain ◽  
Transactivation Activity ◽  
Single Amino Acid Change ◽  
Evolutionarily Conserved

AbstractEcdysoneless (Ecd) is an evolutionarily conserved protein and its function is essential for embryonic development inDrosophilaand cell growth in yeast. However, its function has remained unknown until recently. Studies in yeast suggested a potential role of Ecd in transcription; however, Ecd lacks a DNA-binding domain. Using a GAL4-luciferase reporter assay and a GAL4 DNA-binding domain fusion with Ecd or its mutants, we present evidence that human Ecd has a transactivation activity in its C-terminal region. Importantly, further analyses using point mutants showed that a single amino acid change at either Asp-484 or Leu-489 essentially completely abolishes the transactivation activity of Ecd. We further demonstrate that Ecd interacts with p300, a histone acetyltransferase, and coexpression of Ecd with p300 enhances the Ecd-mediated transactivation activity. Ecd localizes to both nucleus and cytoplasm and shuttles between the nucleus and cytoplasm; however, it exhibits strong nuclear export. Based on previous yeast studies and evidence provided here, we suggest that Ecd functions as a transcriptional regulator. Our results indicate an important function of human Ecd and provide a basis to explore the transcriptional partners of Ecd.

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