An essential yeast gene encoding a TTAGGG repeat-binding protein

1993 ◽  
Vol 13 (2) ◽  
pp. 1306-1314
Author(s):  
C Brigati ◽  
S Kurtz ◽  
D Balderes ◽  
G Vidali ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insertion Mutation ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

scholarly journals An essential yeast gene encoding a TTAGGG repeat-binding protein.

1993 ◽  
Vol 13 (2) ◽  
pp. 1306-1314 ◽  
Author(s):  
C Brigati ◽  
S Kurtz ◽  
D Balderes ◽  
G Vidali ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insertion Mutation ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

scholarly journals Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

2004 ◽  
Vol 3 (2) ◽  
pp. 277-287 ◽  
Author(s):  
Itay Onn ◽  
Neta Milman-Shtepel ◽  
Joseph Shlomai
Keyword(s):  
Dna Binding ◽  
Redox Potential ◽  
Protein Interactions ◽  
Replication Origin ◽  
Binding Protein ◽  
Binding Activity ◽  
Kinetoplast Dna ◽  
Minicircle Sequence

ABSTRACT Kinetoplast DNA, the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region. C. fasciculata trypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.

scholarly journals Identification of the varR Gene as a Transcriptional Regulator of Virginiamycin S Resistance inStreptomyces virginiae

2001 ◽  
Vol 183 (6) ◽  
pp. 2025-2031 ◽  
Author(s):  
Wises Namwat ◽  
Chang-Kwon Lee ◽  
Hiroshi Kinoshita ◽  
Yasuhiro Yamada ◽  
Takuya Nihira
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Northern Blot Analysis ◽  
Binding Activity ◽  
Binding Motif ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Dna Binding Activity ◽  
Streptomyces Virginiae

ABSTRACT A gene designated varR (for virginiaeantibiotic resistance regulator) was identified in Streptomyces virginiae 89 bp downstream of a varS gene encoding a virginiamycin S (VS)-specific transporter. The deduced varRproduct showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the varS promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of varS in a VS-dependent manner. Northern blot analysis revealed that varR was cotranscribed with upstream varS as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of thevarS promoter region clarified two adjacent VarR binding sites in the varS promoter.

scholarly journals The U3 small nucleolar ribonucleoprotein component Imp4p is a telomeric DNA-binding protein

2007 ◽  
Vol 408 (3) ◽  
pp. 387-393 ◽  
Author(s):  
Yi-Ching Hsieh ◽  
Pei-Jung Tu ◽  
Ying-Yuan Lee ◽  
Chun-Chen Kuo ◽  
Yi-Chien Lin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Mobility Shift ◽  
Telomere Function

Imp4p is a component of U3 snoRNP (small nucleolar ribonucleoprotein) involved in the maturation of 18S rRNA. We have shown that Imp4p interacts with Cdc13p, a single-stranded telomere-binding protein involved in telomere maintenance. To understand the role of Imp4p in telomeres, we purified recombinant Imp4p protein and tested its binding activity towards telomeric DNA using electrophoretic mobility-shift assays. Our results showed that Imp4p bound specifically to single-stranded telomeric DNA in vitro. The interaction of Imp4p to telomeres in vivo was also demonstrated by chromatin immunoprecipitation experiments. Significantly, the binding of Imp4p to telomeres was not limited to yeast proteins, since the hImp4 (human Imp4) also bound to vertebrate single-stranded telomeric DNA. Thus we conclude that Imp4p is a novel telomeric DNA-binding protein that, in addition to its role in rRNA processing, might participate in telomere function.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation

2001 ◽  
Vol 29 (6) ◽  
pp. 688-691 ◽  
Author(s):  
K. J. Campbell ◽  
N. R. Chapman ◽  
N. D. Perkins
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Uv Light ◽  
Binding Protein ◽  
Nuclear Factor Κb ◽  
Camp Response Element Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Dna Binding Activity

The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes

1989 ◽  
Vol 9 (5) ◽  
pp. 2018-2024
Author(s):  
D L Johnson ◽  
S L Wilson
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Iii ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Trna Genes ◽  
Dna Binding Activity ◽  
Transcription In Vitro ◽  
Affinity Interaction ◽  
Dna Complex

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.

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