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 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 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.

scholarly journals Cell surface recycling in yeast: mechanisms and machineries

2016 ◽  
Vol 44 (2) ◽  
pp. 474-478 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Genetic System ◽  
Integral Membrane Protein ◽  
Protein Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif

1993 ◽  
Vol 13 (3) ◽  
pp. 1572-1582
Author(s):  
P Dobrzanski ◽  
R P Ryseck ◽  
R Bravo
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Wild Type ◽  
Nf Kappa B ◽  
Hybrid Molecules ◽  
In Cis ◽  
Rel Homology Domain

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

scholarly journals Both N- and C-terminal domains of RelB are required for full transactivation: role of the N-terminal leucine zipper-like motif.

1993 ◽  
Vol 13 (3) ◽  
pp. 1572-1582 ◽  
Author(s):  
P Dobrzanski ◽  
R P Ryseck ◽  
R Bravo
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Wild Type ◽  
Nf Kappa B ◽  
Hybrid Molecules ◽  
In Cis ◽  
Rel Homology Domain

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.

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 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.

Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 442-451
Author(s):  
M Nishizawa ◽  
R Araki ◽  
Y Teranishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Transcriptional Activation ◽  
Regulatory Elements ◽  
Noncoding Region ◽  
Yeast Cells ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

scholarly journals Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae

Microbiology ◽  
2011 ◽  
Vol 157 (3) ◽  
pp. 879-889 ◽  
Author(s):  
Hugo Hernández ◽  
Cristina Aranda ◽  
Geovani López ◽  
Lina Riego ◽  
Alicia González
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Specific Binding ◽  
Transcriptional Response ◽  
Transcriptional Regulator ◽  
Transcriptional Responses ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Transcriptional Complex ◽  
Nitrogen Conditions

The transcriptional activation response relies on a repertoire of transcriptional activators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. We have addressed the possibility of achieving novel transcriptional responses by the construction of a new transcriptional regulator – the Hap2-3-5-Gln3 hybrid modulator – harbouring the HAP complex polypeptides that constitute the DNA-binding domain (Hap2-3-5) and the Gln3 activation domain, which usually act in an uncombined fashion. The results presented in this paper show that transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions is achieved through the action of the novel Hap2-3-5-Gln3 transcriptional regulator. We propose that the combination of the Hap DNA-binding and Gln3 activation domains results in a hybrid modulator that elicits a novel transcriptional response not evoked when these modulators act independently.

scholarly journals Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus

2002 ◽  
Vol 13 (2) ◽  
pp. 670-682 ◽  
Author(s):  
Steven M. Markus ◽  
Samir S. Taneja ◽  
Susan K. Logan ◽  
Wenhui Li ◽  
Susan Ha ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Hybrid Approach ◽  
Independent Manner ◽  
Activation Domains ◽  
Cell Library ◽  
Nuclear Extracts ◽  
N Terminus ◽  
Transcriptional Activation Domains

The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR153–336, containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR153–336 fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.

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