Equilibrium Analysis of the DNA Binding Domain of the Ultraspiracle Protein Interaction with the Response Element from the hsp27 Gene Promoter—the Application of Molecular Beacon Technology

2007 ◽  
Vol 18 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Tomasz Krusiński ◽  
Marta Wietrzych ◽  
Iwona Grad ◽  
Andrzej Ożyhar ◽  
Piotr Dobryszycki
Keyword(s):  
Dna Binding ◽  
Protein Interaction ◽  
Molecular Beacon ◽  
Gene Promoter ◽  
Binding Domain ◽  
Equilibrium Analysis ◽  
Dna Binding Domain ◽  
Response Element

scholarly journals Two domains of ISGF3 gamma that mediate protein-DNA and protein-protein interactions during transcription factor assembly contribute to DNA-binding specificity.

1993 ◽  
Vol 13 (1) ◽  
pp. 196-206 ◽  
Author(s):  
S A Veals ◽  
T Santa Maria ◽  
D E Levy
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interaction ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Response Element ◽  
Ifn Alpha ◽  
Dna Binding Specificity

Alpha interferon (IFN-alpha) induces the transcription of a large set of genes through activation of multimeric transcription factor ISGF3. This factor can be dissociated into two protein components, termed ISGF3 gamma and ISGF3 alpha. ISGF3 gamma is a 48-kDa protein related at the amino terminus to members of the IFN-regulatory factor (IRF) and Myb families of DNA-binding proteins; ISGF3 alpha consists of three polypeptides of 84, 91, and 113 kDa that self-assemble to form an activated component in response to IFN-alpha. DNA-binding studies indicated that ISGF3 gamma binds DNA alone, recognizing the IFN-stimulated response element, while the ISGF3 alpha polypeptides alone display no specific interactions with DNA. A complex between ISGF3 gamma and activated ISGF3 alpha binds the IFN-stimulated response element with much greater affinity than does the 48-kDa ISGF3 gamma protein alone. The DNA-binding domain of ISGF3 gamma and regions responsible for protein-protein interaction with ISGF3 alpha were identified by using deleted forms of ISGF3 gamma expressed in vitro. The amino-terminal region of ISGF3 gamma homologous to the IRF and Myb proteins was sufficient for interaction with DNA and displayed the binding specificity of the intact protein; phosphorylation of this region was necessary for activity. A second region of 160 amino acids separated from the DNA-binding domain by over 100 amino acids contained a domain capable of associating with ISGF3 alpha and was sufficient to confer specific ISGF3 alpha interaction to a heterologous protein. Interaction of the ISGF3 alpha component with the protein interaction domain of ISGF3 gamma altered the DNA-binding specificity of the resulting complex, suggesting that one or more of the ISGF3 alpha polypeptides make base-specific contacts with DNA. This interaction defines a mechanism through which IRF-like proteins complexed with regulatory components can display novel DNA-binding specificities.

Two domains of ISGF3 gamma that mediate protein-DNA and protein-protein interactions during transcription factor assembly contribute to DNA-binding specificity

1993 ◽  
Vol 13 (1) ◽  
pp. 196-206
Author(s):  
S A Veals ◽  
T Santa Maria ◽  
D E Levy
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interaction ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Response Element ◽  
Ifn Alpha ◽  
Dna Binding Specificity

Alpha interferon (IFN-alpha) induces the transcription of a large set of genes through activation of multimeric transcription factor ISGF3. This factor can be dissociated into two protein components, termed ISGF3 gamma and ISGF3 alpha. ISGF3 gamma is a 48-kDa protein related at the amino terminus to members of the IFN-regulatory factor (IRF) and Myb families of DNA-binding proteins; ISGF3 alpha consists of three polypeptides of 84, 91, and 113 kDa that self-assemble to form an activated component in response to IFN-alpha. DNA-binding studies indicated that ISGF3 gamma binds DNA alone, recognizing the IFN-stimulated response element, while the ISGF3 alpha polypeptides alone display no specific interactions with DNA. A complex between ISGF3 gamma and activated ISGF3 alpha binds the IFN-stimulated response element with much greater affinity than does the 48-kDa ISGF3 gamma protein alone. The DNA-binding domain of ISGF3 gamma and regions responsible for protein-protein interaction with ISGF3 alpha were identified by using deleted forms of ISGF3 gamma expressed in vitro. The amino-terminal region of ISGF3 gamma homologous to the IRF and Myb proteins was sufficient for interaction with DNA and displayed the binding specificity of the intact protein; phosphorylation of this region was necessary for activity. A second region of 160 amino acids separated from the DNA-binding domain by over 100 amino acids contained a domain capable of associating with ISGF3 alpha and was sufficient to confer specific ISGF3 alpha interaction to a heterologous protein. Interaction of the ISGF3 alpha component with the protein interaction domain of ISGF3 gamma altered the DNA-binding specificity of the resulting complex, suggesting that one or more of the ISGF3 alpha polypeptides make base-specific contacts with DNA. This interaction defines a mechanism through which IRF-like proteins complexed with regulatory components can display novel DNA-binding specificities.

scholarly journals BZLF1 Activation of the Methylated Form of the BRLF1 Immediate-Early Promoter Is Regulated by BZLF1 Residue 186

2005 ◽  
Vol 79 (12) ◽  
pp. 7338-7348 ◽  
Author(s):  
Prasanna M. Bhende ◽  
William T. Seaman ◽  
Henri-Jacques Delecluse ◽  
Shannon C. Kenney
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Viral Genome ◽  
Gene Promoter ◽  
Binding Domain ◽  
Immediate Early ◽  
Dna Binding Domain ◽  
Barr Virus ◽  
Z Dna ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the BRLF1 IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending

1992 ◽  
Vol 12 (5) ◽  
pp. 2037-2042
Author(s):  
A M Nardulli ◽  
D J Shapiro
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Dna Bending ◽  
Binding Domain ◽  
Estrogen Response Element ◽  
Dna Binding Domain ◽  
Mobility Shift ◽  
Response Element ◽  
Estrogen Response ◽  
Dna Fragment

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

scholarly journals Functional Cross-Antagonism between Transcription Factors FLI-1 and EKLF

2003 ◽  
Vol 23 (4) ◽  
pp. 1390-1402 ◽  
Author(s):  
Joëlle Starck ◽  
Nathalie Cohet ◽  
Colette Gonnet ◽  
Sandrine Sarrazin ◽  
Zina Doubeikovskaia ◽  
...  
Keyword(s):  
Dna Binding ◽  
Globin Gene ◽  
Gene Promoter ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Protein Interaction ◽  
Repressive Effect ◽  
Ets Family ◽  
Ets Domain ◽  
Repression Domain

ABSTRACT FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the β-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on β-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.

scholarly journals DNA recognition by the androgen receptor: evidence for an alternative DNA-dependent dimerization, and an active role of sequences flanking the response element on transactivation

2003 ◽  
Vol 369 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Annemie HAELENS ◽  
Guy VERRIJDT ◽  
Leen CALLEWAERT ◽  
Valerie CHRISTIAENS ◽  
Kris SCHAUWAERS ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Dna Binding ◽  
Steroid Receptors ◽  
Binding Domain ◽  
Hinge Region ◽  
General Mechanism ◽  
Affinity Binding ◽  
Dna Binding Domain ◽  
Direct Repeats ◽  
Response Element

The androgen receptor has a subset of target DNA sequences, which are not recognized by any other steroid receptors. The androgen selectivity of these sequences was proposed to be the consequence of the ability of the androgen receptor to dimerize on direct repeats of 5′-TGTTCT-3′-like sequences. This is in contrast with the classical non-selective elements consisting of inverted repeats of the 5′-TGTTCT-3′ elements separated by three nucleotides and which are recognized by other steroid receptors in addition to the androgen receptor. We demonstrate that while the DNA-binding domain of the oestrogen receptor is unable to dimerize on direct repeats, dimeric binding can be rescued by replacing the second Zn finger and part of the hinge region by the corresponding fragment of the androgen receptor, but not the glucocorticoid receptor. In this study, we investigate the androgen receptor binding to all natural androgen-selective response elements described so far. We show that a 12-amino acid C-terminal extension of the DNA-binding domain is required for high-affinity binding of the androgen receptor to all these elements. For one androgen-specific low-affinity binding site, the flanking sequences do not contribute to the invitro affinity of the androgen receptor DNA-binding domain. Surprisingly, however, they control the transcriptional activity of the androgen receptor in transient transfection experiments. In conclusion, we give evidence that the alternative DNA-dependent dimerization of the androgen receptor on direct repeats is a general mechanism for androgen specificity in which the second Zn finger and hinge region are involved. In addition, the sequences flanking an androgen-response element can control the activity of the androgen receptor.

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