scholarly journals Identification, Mutational Analysis, and Coactivator Requirements of Two Distinct Transcriptional Activation Domains of the Saccharomyces cerevisiae Hap4 Protein

2004 ◽  
Vol 3 (2) ◽  
pp. 339-347 ◽  
Author(s):  
John L. Stebbins ◽  
Steven J. Triezenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Mutational Analysis ◽  
Transcriptional Coactivator ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Activation Domains

ABSTRACT The Hap4 protein of the budding yeast Saccharomyces cerevisiae activates the transcription of genes that are required for growth on nonfermentable carbon sources. Previous reports suggested the presence of a transcriptional activation domain within the carboxyl-terminal half of Hap4 that can function in the absence of Gcn5, a transcriptional coactivator protein and histone acetyltransferase. The boundaries of this activation domain were further defined to a region encompassing amino acids 359 to 476. Within this region, several clusters of hydrophobic amino acids are critical for transcriptional activity. This activity does not require GCN5 or two other components of the SAGA coactivator complex, SPT3 and SPT8, but it does require SPT7 and SPT20. Contrary to previous reports, a Hap4 fragment comprising amino acids 1 to 330 can support the growth of yeast on lactate medium, and when tethered to lexA, can activate a reporter gene with upstream lexA binding sites, demonstrating the presence of a second transcriptional activation domain. In contrast to the C-terminal activation domain, the transcriptional activity of this N-terminal region depends on GCN5. We conclude that the yeast Hap4 protein has at least two transcriptional activation domains with strikingly different levels of dependence on specific transcriptional coactivator proteins.

scholarly journals The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress.

1996 ◽  
Vol 16 (3) ◽  
pp. 839-846 ◽  
Author(s):  
E M Newton ◽  
U Knauf ◽  
M Green ◽  
R E Kingston
Keyword(s):  
Amino Acids ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Acid Activation ◽  
Regulatory Domain ◽  
Activation Domain ◽  
Control Temperature ◽  
Activation Domains ◽  
Transcriptional Activation Domains

Heat shock factor (HSF) activates transcription in response to cellular stress. Human HSF1 has a central regulatory domain which can repress the activity of its activation domains at the control temperature and render them heat shock inducible. To determine whether the regulatory domain works in tandem with specific features of the HSF1 transcriptional activation domains, we first used deletion and point mutagenesis to define these activation domains. One of the activation domains can be reduced to just 20 amino acids. A GAL4 fusion protein containing the HSF 1 regulatory domain and this 20-amino-acid activation domain is repressed at the control temperature but potently activates transcription in response to heat shock. No specific amino acids in this activation domain are required for response to the regulatory domain; in particular, none of the potentially phosphorylated serine and threonine residues are required for heat induction, implying that heat-induced phosphorylation of the transcriptional activation domains is not required for induction. The regulatory domain is able to confer heat responsiveness to an otherwise completely heterologous chimeric activator that contains a portion of the VP16 activation domain, suggesting that the regulatory domain can sense heat in the absence of other portions of HSF1.

scholarly journals Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535 ◽  
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

scholarly journals Transcriptional activation upon pheromone stimulation mediated by a small domain of Saccharomyces cerevisiae Ste12p.

1997 ◽  
Vol 17 (11) ◽  
pp. 6410-6418 ◽  
Author(s):  
H Pi ◽  
C T Chien ◽  
S Fields
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Activation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Hybrid Proteins ◽  
High Level ◽  
Transcriptional Induction

In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element. We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins. A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity. Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation. By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction. This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone. By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines. The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain. These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain.

Fos and jun cooperate in transcriptional regulation via heterologous activation domains

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

scholarly journals Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains

1998 ◽  
Vol 329 (2) ◽  
pp. 395-403 ◽  
Author(s):  
Gianluca TELL ◽  
Lorena PERRONE ◽  
Dora FABBRO ◽  
Lucia PELLIZZARI ◽  
Carlo PUCILLO ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Acidic Domain ◽  
Thyroid Transcription Factor 1 ◽  
Thyroid Transcription Factor ◽  
Transcriptional Activation Domains ◽  
Transcription Factor 1

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of α-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into α-helices or β-strands can be induced. Therefore our data indicate that the inducibility of α-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

scholarly journals MLL and CREB Bind Cooperatively to the Nuclear Coactivator CREB-Binding Protein

2001 ◽  
Vol 21 (7) ◽  
pp. 2249-2258 ◽  
Author(s):  
Patricia Ernst ◽  
Jing Wang ◽  
Mary Huang ◽  
Richard H. Goodman ◽  
Stanley J. Korsmeyer
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Single Amino Acid ◽  
Creb Binding Protein ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Adenovirus E1a

ABSTRACT A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB–CREB-binding protein (CBP) complex. When tested for binding to CREB or CBP individually, this MLL fragment interacted directly with CBP, but not with CREB. In vitro binding experiments refined the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain). The transactivation activity of MLL was dependent on CBP, as either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction proved effective at inhibiting MLL-mediated transactivation. Single amino acid substitutions within the MLL activation domain revealed that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, were critical for the interaction of MLL with CBP. Using purified components, we found that the MLL activation domain facilitated the binding of CBP to phosphorylated CREB. In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP.

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 Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

scholarly journals Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1666-1674 ◽  
Author(s):  
P A Moore ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Transcriptional Activator ◽  
Genetic System ◽  
Nf Kappa B ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Inhibitory Molecule

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.

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.

Sign in / Sign up

Export Citation Format

Share Document