scholarly journals B-Cell Coactivator OBF-1 Exhibits Unusual Transcriptional Properties and Functions in a DNA-Bound Oct-1-Dependent Fashion

1999 ◽  
Vol 19 (6) ◽  
pp. 4247-4254 ◽  
Author(s):  
Andrea Krapp ◽  
Michel Strubin
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Regulatory Elements ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains

ABSTRACT Eukaryotic transcriptional activators generally comprise both a DNA-binding domain that recognizes specific cis-regulatory elements in the target genes and an activation domain which is essential for transcriptional stimulation. Activation domains typically behave as structurally and functionally autonomous modules that retain their intrinsic activities when directed to a promoter by a variety of heterologous DNA-binding domains. Here we report that OBF-1, a B-cell-specific coactivator for transcription factor Oct-1, challenges this traditional view in that it contains an atypical activation domain that exhibits two unexpected functional properties when tested in the yeast Saccharomyces cerevisiae. First, OBF-1 by itself has essentially no intrinsic activation potential, yet it strongly synergizes with other activation domains such as VP16 and Gal4. Second, OBF-1 exerts its effect in association with DNA-bound Oct-1 but is inactive when attached to a heterologous DNA-binding domain. These findings suggest that activation by OBF-1 is not obtained by simple recruitment of the coactivator to the promoter but requires interaction with DNA-bound Oct-1 to stimulate a step distinct from those regulated by classical activation domains.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Structure of the XPA DNA binding domain and RPA high-affinity DNA binding domains on a model NER substrate

2019 ◽  
Vol 75 (a1) ◽  
pp. a203-a203
Author(s):  
Walter J. Chazin ◽  
Agnieszka M. Topolska-Woś ◽  
Norie Sugitani ◽  
John J. Cordoba ◽  
Hyun Suk Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains

scholarly journals A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function.

1995 ◽  
Vol 15 (6) ◽  
pp. 3354-3362 ◽  
Author(s):  
M Green ◽  
T J Schuetz ◽  
E K Sullivan ◽  
R E Kingston
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Chimeric Proteins ◽  
Dna Binding Domain ◽  
Regulatory Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domains

Human heat shock factor 1 (HSF1) stimulates transcription from heat shock protein genes following stress. We have used chimeric proteins containing the GAL4 DNA binding domain to identify the transcriptional activation domains of HSF1 and a separate domain that is capable of regulating activation domain function. This regulatory domain conferred heat shock inducibility to chimeric proteins containing the activation domains. The regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3. The regulatory domain was found to be functionally homologous between chicken and human HSF1. This domain does not affect DNA binding by the chimeric proteins and does not contain any of the sequences previously postulated to regulate DNA binding of HSF1. Thus, we suggest that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.

scholarly journals Specific Binding of Integrase to the Origin of Transfer (oriT) of the Conjugative Transposon Tn916

2001 ◽  
Vol 183 (9) ◽  
pp. 2947-2951 ◽  
Author(s):  
Douglas Hinerfeld ◽  
Gordon Churchward
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Conjugal Transfer ◽  
Dna Binding Domain ◽  
Conjugative Transposon ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
A Site ◽  
Integrase Protein

ABSTRACT Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding tooriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.

scholarly journals Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a σ54-Dependent Transcriptional Activator

2004 ◽  
Vol 186 (11) ◽  
pp. 3499-3507 ◽  
Author(s):  
Hao Xu ◽  
Baohua Gu ◽  
B. Tracy Nixon ◽  
Timothy R. Hoover
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Sinorhizobium Meliloti ◽  
Atp Hydrolysis ◽  
Binding Domain ◽  
Stable Complex ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
Binding Domains

ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD( Δ 1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD( Δ 1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD( Δ 1-142) displayed multiple species. The DctD AAA+ domain, but not DctD( Δ 1-142), formed a stable complex with σ54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54 in addition to its role in recognition of upstream activation sequences.

scholarly journals The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.

1992 ◽  
Vol 12 (1) ◽  
pp. 266-275 ◽  
Author(s):  
J J Schwarz ◽  
T Chakraborty ◽  
J Martin ◽  
J M Zhou ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
E Box ◽  
Myogenic Program ◽  
Basic Region

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

scholarly journals An Engineered PAX3-KRAB Transcriptional Repressor Inhibits the Malignant Phenotype of Alveolar Rhabdomyosarcoma Cells Harboring the Endogenous PAX3-FKHR Oncogene

2000 ◽  
Vol 20 (14) ◽  
pp. 5019-5031 ◽  
Author(s):  
William J. Fredericks ◽  
Kasirajan Ayyanathan ◽  
Meenhard Herlyn ◽  
Josh R. Friedman ◽  
Frank J. Rauscher
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Binding Domain ◽  
Stable Expression ◽  
Alveolar Rhabdomyosarcoma ◽  
Dna Binding Domain ◽  
Malignant Phenotype ◽  
Activation Domain

ABSTRACT The t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma tumors (ARMS) creates an oncogenic transcriptional activator by fusion of PAX3 DNA binding motifs to a COOH-terminal activation domain derived from the FKHR gene. The dominant oncogenic potential of the PAX3-FKHR fusion protein is dependent on the FKHR activation domain. We have fused the KRAB repression module to the PAX3 DNA binding domain as a strategy to suppress the activity of the PAX3-FKHR oncogene. The PAX3-KRAB protein bound PAX3 target DNA sequences and repressed PAX3-dependent reporter plasmids. Stable expression of the PAX3-KRAB protein in ARMS cell lines resulted in loss of the ability of the cells to grow in low-serum or soft agar and to form tumors in SCID mice. Stable expression of a PAX3-KRAB mutant, which lacks repression function, or a KRAB protein alone, lacking a PAX3 DNA binding domain, failed to suppress the ARMS malignant phenotype. These data suggest that the PAX3-KRAB repressor functions as a DNA-binding-dependent suppressor of the transformed phenotype of ARMS cells, probably via competition with the endogenous PAX3-FKHR oncogene and repression of target genes required for ARMS tumorigenesis. The engineered repressor approach that directs a transcriptional repression domain to target genes deregulated by the PAX3-FKHR oncogene may be a useful strategy to identify the target genes critical for ARMS tumorigenesis.

scholarly journals Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.

1996 ◽  
Vol 16 (3) ◽  
pp. 792-799 ◽  
Author(s):  
S L Gregory ◽  
R D Kortschak ◽  
B Kalionis ◽  
R Saint
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
The Dead

We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.

scholarly journals Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from drosophila melanogaster.

1996 ◽  
Vol 43 (4) ◽  
pp. 611-621 ◽  
Author(s):  
A Rusin ◽  
A Niedziela-Majka ◽  
G Rymarczyk ◽  
A Ozyhar
Keyword(s):  
Dna Binding ◽  
Dna Interaction ◽  
Binding Domain ◽  
Structural Basis ◽  
Dna Binding Domain ◽  
Affinity Tag ◽  
Mobility Shift ◽  
Ecdysteroid Receptor ◽  
Dna Binding Domains ◽  
Binding Domains

Two members of the nuclear receptor superfamily, EcR and Ultraspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone-the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development. In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains. Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30. Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16%. Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose. The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene.

scholarly journals Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067 ◽  
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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