scholarly journals τ91, an Essential Subunit of Yeast Transcription Factor IIIC, Cooperates with τ138 in DNA Binding

1998 ◽  
Vol 18 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Rosalía Arrebola ◽  
Nathalie Manaud ◽  
Sophie Rozenfeld ◽  
Marie-Claude Marsolier ◽  
Olivier Lefebvre ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Essential Gene ◽  
Open Reading Frames ◽  
Class Iii ◽  
Gene Encoding ◽  
Promoter Recognition

ABSTRACT Transcription factor IIIC (TFIIIC) (or τ) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, τ91, present in affinity-purified TFIIIC. τ91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of τ91 was established by the following biochemical and genetic evidence. Antibodies to τ91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to τ91; and a point mutation in TFC6 (τ91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349Emutant affected in the B block binding subunit (τ138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that τ91 cooperates with τ138 for DNA binding. Recombinant τ91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

scholarly journals Characterization of the Pasteurella multocida hgbA Gene Encoding a Hemoglobin-Binding Protein

2002 ◽  
Vol 70 (11) ◽  
pp. 5955-5964 ◽  
Author(s):  
Montserrat Bosch ◽  
M. Elena Garrido ◽  
Montserrat Llagostera ◽  
Ana M. Pérez de Rozas ◽  
Ignacio Badiola ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Bacterial Pathogen ◽  
Open Reading Frames ◽  
Transcriptional Unit ◽  
Gene Encoding ◽  
Hemoglobin Binding ◽  
Reading Frames

ABSTRACT Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.

scholarly journals A Chimeric Subunit of Yeast Transcription Factor IIIC Forms a Subcomplex with τ95

1998 ◽  
Vol 18 (6) ◽  
pp. 3191-3200 ◽  
Author(s):  
Nathalie Manaud ◽  
Rosalía Arrebola ◽  
Bénédicte Buffin-Meyer ◽  
Olivier Lefebvre ◽  
Hartmut Voss ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Essential Gene ◽  
Cell Metabolism ◽  
Chimeric Protein ◽  
Gene Encoding ◽  
Multifunctional Protein ◽  
Isolation And Characterization ◽  
Promoter Recognition ◽  
Transcription Factor Iiib

ABSTRACT The multisubunit yeast transcription factor IIIC (TFIIIC) is a multifunctional protein required for promoter recognition, transcription factor IIIB recruitment, and chromatin antirepression. We report the isolation and characterization of TFC7, an essential gene encoding the 55-kDa polypeptide, τ55, present in affinity-purified TFIIIC. τ55 is a chimeric protein generated by an ancient chromosomal rearrangement. Its C-terminal half is essential for cell viability and sufficient to ensure TFIIIC function in DNA binding and transcription assays. The N-terminal half is nonessential and highly similar to a putative yeast protein encoded on another chromosome and to a cyanobacterial protein of unknown function. Partial deletions of the N-terminal domain impaired τ55 function at a high temperature or in media containing glycerol or ethanol, suggesting a link between PolIII transcription and metabolic pathways. Interestingly, τ55 was found, together with TFIIIC subunit τ95, in a protein complex which was distinct from TFIIIC and which may play a role in the regulation of PolIII transcription, possibly in relation to cell metabolism.

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.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1259-1268 ◽  
Author(s):  
A. Meng ◽  
B. Moore ◽  
H. Tang ◽  
B. Yuan ◽  
S. Lin
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Dna Binding Domain ◽  
Related Gene ◽  
Presomitic Mesoderm ◽  
Zinc Finger Gene ◽  
Human And Mouse

The Drosophila doublesex (dsx) gene encodes a transcription factor that mediates sex determination. We describe the characterization of a novel zebrafish zinc-finger gene, terra, which contains a DNA binding domain similar to that of the Drosophila dsx gene. However, unlike dsx, terra is transiently expressed in the presomitic mesoderm and newly formed somites. Expression of terra in presomitic mesoderm is restricted to cells that lack expression of MyoD. In vivo, terra expression is reduced by hedgehog but enhanced by BMP signals. Overexpression of terra induces rapid apoptosis both in vitro and in vivo, suggesting that a tight regulation of terra expression is required during embryogenesis. Terra has both human and mouse homologs and is specifically expressed in mouse somites. Taken together, our findings suggest that terra is a highly conserved protein that plays specific roles in early somitogenesis of vertebrates.

scholarly journals Cystine/glutamate antiporter xCT (SLC7A11) facilitates oncogenic RAS transformation by preserving intracellular redox balance

2019 ◽  
Vol 116 (19) ◽  
pp. 9433-9442 ◽  
Author(s):  
Jonathan K. M. Lim ◽  
Alberto Delaidelli ◽  
Sean W. Minaker ◽  
Hai-Feng Zhang ◽  
Milena Colovic ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Redox Balance ◽  
Gene Encoding ◽  
Oncogenic Ras ◽  
Opposing Effects ◽  
Whole Transcriptome ◽  
Intracellular Redox

The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach, we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo by mitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.

scholarly journals Characterization of Endogenous Retroviruses in Sheep

2003 ◽  
Vol 77 (20) ◽  
pp. 11268-11273 ◽  
Author(s):  
Nikolai Klymiuk ◽  
Mathias Müller ◽  
Gottfried Brem ◽  
Bernhard Aigner
Keyword(s):  
Endogenous Retrovirus ◽  
Ovis Aries ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
In Vivo Experiments ◽  
Pregnant Sheep ◽  
Reading Frames

ABSTRACT Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

scholarly journals Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer

2012 ◽  
Vol 68 (11) ◽  
pp. 1354-1358 ◽  
Author(s):  
Marie V. Wong ◽  
Paaventhan Palasingam ◽  
Prasanna R. Kolatkar
Keyword(s):  
Dna Binding ◽  
Potassium Acetate ◽  
Class Iii ◽  
Data Set ◽  
Cell Parameters ◽  
Helix Loop Helix ◽  
Id Protein ◽  
Bhlh Transcription Factors

The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix–loop–helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-livesin vivoand reportedin vitroinstability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1 Å resolution data set was collected from a crystal belonging to space groupP3121 with unit-cell parametersa=b= 51.622,c= 111.474 Å, α = β = 90, γ = 120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1 MMES pH 6.5, 2.0 Mpotassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.

scholarly journals TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1

Blood ◽  
2017 ◽  
Vol 129 (10) ◽  
pp. 1284-1295 ◽  
Author(s):  
Lorenz Jahn ◽  
Pleun Hombrink ◽  
Renate S. Hagedoorn ◽  
Michel G. D. Kester ◽  
Dirk M. van der Steen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
B Cell ◽  
B Cell Malignancies ◽  
High Affinity ◽  
Isolation And Characterization ◽  
Key Points

Key Points Isolation and characterization of a high-affinity TCR targeting the intracellular B cell–specific transcription factor BOB1. T cells expressing a BOB1-specific TCR lysed and eradicated primary multiple myeloma and other B-cell malignancies in vitro and in vivo.

scholarly journals Constitutive nuclear NFκB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate

1995 ◽  
Vol 312 (3) ◽  
pp. 833-838 ◽  
Author(s):  
A F G Slater ◽  
M Kimland ◽  
S A Jiang ◽  
S Orrenius
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Gradient Centrifugation ◽  
Binding Activity ◽  
Pyrrolidine Dithiocarbamate ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Apoptotic Activity

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

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