scholarly journals The scs' boundary element: characterization of boundary element-associated factors.

1997 ◽  
Vol 17 (2) ◽  
pp. 999-1009 ◽  
Author(s):  
C M Hart ◽  
K Zhao ◽  
U K Laemmli
Keyword(s):  
Dna Binding ◽  
Boundary Element ◽  
Target Genes ◽  
Polytene Chromosomes ◽  
Amino Terminal ◽  
High Affinity ◽  
Dna Binding Specificity ◽  
Carboxy Terminal ◽  
Terminal Domains

Boundary elements are thought to define the peripheries of chromatin domains and to restrict enhancer-promoter interactions to their target genes within their domains. We previously characterized a cDNA encoding the BEAF-32A protein (32A), which binds with high affinity to the scs' boundary element from the Drosophila melanogaster 87A7 hsp70 locus. Here, we report a second protein, BEAF-32B, that differs from 32A only in its amino terminus. Unlike 32A, it has the same DNA binding specificity as the complete BEAF activity affinity purified from Drosophila. We characterize three domains in these proteins. Heterocomplex formation is mediated by their identical carboxy-terminal domains, and DNA binding is mediated by their unique amino-terminal domains. The identical middle domains of 32A and 32B are dispensable for the functions described here, although they may be important for boundary element function. 32A and 32B apparently form trimers, and the ratio of 32A to 32B varies at different loci on polytene chromosomes as judged by immunofluorescence. The scs' element contains a high- and low-affinity binding site for BEAF. We observed that interaction with the low-affinity site is facilitated by binding to the high-affinity site some 200 bp distant.

scholarly journals Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor.

1995 ◽  
Vol 15 (5) ◽  
pp. 2517-2526 ◽  
Author(s):  
V Giguère ◽  
L D McBroom ◽  
G Flock
Keyword(s):  
Dna Binding ◽  
Nuclear Receptors ◽  
Target Gene ◽  
Binding Studies ◽  
Orphan Nuclear Receptors ◽  
Retinoid Receptor ◽  
Amino Terminal ◽  
High Affinity ◽  
Carboxy Terminal ◽  
Terminal Domains

The ROR alpha isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct amino-terminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR alpha 1 isoform and have now identified the minimal sequence both in its amino- and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.

scholarly journals Identification of a DNA Binding Region in GerE fromBacillus subtilis

2001 ◽  
Vol 183 (14) ◽  
pp. 4183-4189 ◽  
Author(s):  
Dinene L. Crater ◽  
Charles P. Moran
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Small Region ◽  
Wild Type ◽  
Binding Region ◽  
Activator Proteins ◽  
Amino Terminal ◽  
High Affinity ◽  
Carboxy Terminal

ABSTRACT Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins. Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins. GerE fromBacillus subtilis is the smallest member of the LuxR-FixJ family. Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif. Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding. Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA. We examined the effects of single base pair substitutions in the high-affinity GerE binding site on thesigK promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on thesigK promoter are important for a high-affinity interaction with GerE. We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites. We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites. These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.

scholarly journals Further Characterization of Functional Domains of PerA, Role of Amino and Carboxy Terminal Domains in DNA Binding

PLoS ONE ◽  
2013 ◽  
Vol 8 (2) ◽  
pp. e56977 ◽  
Author(s):  
J. Antonio Ibarra ◽  
Claudia M. García-Zacarias ◽  
Cristina Lara-Ochoa ◽  
Alejandro Carabarin-Lima ◽  
J. Sergio Tecpanecatl-Xihuitl ◽  
...  
Keyword(s):  
Dna Binding ◽  
Functional Domains ◽  
Carboxy Terminal ◽  
Terminal Domains

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

scholarly journals Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

2003 ◽  
Vol 17 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Raj Kumar ◽  
E. Brad Thompson
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Hormone Receptors ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Protein Interactions ◽  
Carboxy Terminal ◽  
And Function ◽  
Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1

1992 ◽  
Vol 12 (10) ◽  
pp. 4297-4304 ◽  
Author(s):  
D Ray ◽  
R Bosselut ◽  
J Ghysdael ◽  
M G Mattei ◽  
A Tavitian ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
New Gene ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hematopoietic Cell Lines

We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.

Expression and characterization of von Willebrand factor dimerization defects in different types of von Willebrand disease

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 2059-2066 ◽  
Author(s):  
Reinhard Schneppenheim ◽  
Ulrich Budde ◽  
Tobias Obser ◽  
Jacqueline Brassard ◽  
Kerstin Mainusch ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Recombinant Expression ◽  
Von Willebrand Disease ◽  
Disulfide Bonding ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Dimerization defects of von Willebrand factor (vWF) protomers underlie von Willebrand disease (vWD) type 2A, subtype IID (vWD 2A/IID), and corresponding mutations have been identified at the 3′ end of the vWF gene in exon 52. This study identified and expressed 2 additional mutations in this region, a homozygous defect in a patient with vWD type 3 (C2754W) and a heterozygous frameshift mutation (8566delC) in a patient with vWD type 2A, subtype IIE. Both mutations involve cysteine residues that we propose are possibly essential for dimerization. To prove this hypothesis, transient recombinant expression of each of the 2 mutations introduced in the carboxy-terminal vWF fragment II and in the complete vWF complementary DNA, respectively, were carried out in COS-7 cells and compared with expression of vWD 2A/IID mutation C2773R and the wild-type (WT) sequence in COS-7 cells. Recombinant WT vWF fragment II assembled correctly into a dimer, whereas recombinant mutant fragments were monomeric. Homozygous expression of recombinant mutant full-length vWF resulted in additional dimers, probably through disulfide bonding at the amino-terminal multimerization site, whereas recombinant WT vWF correctly assembled into multimers. Coexpression of recombinant mutant and recombinant WT vWF reproduced the multimer patterns observed in heterozygous individuals. Our results suggest that a common defect of vWF biosynthesis—lack of vWF dimerization—may cause diverse types and subtypes of vWD. We also confirmed previous studies that found that disulfide bonding at the vWF amino-terminal is independent of dimerization at the vWF carboxy-terminal.

Sign in / Sign up

Export Citation Format

Share Document