A 40-kDa NF1-like protein, not YY1, binds to the rat p53 promoter for transactivation in various rat organs

1999 ◽  
Vol 77 (3) ◽  
pp. 209-214 ◽  
Author(s):  
Minhyung Lee ◽  
Haisun Song ◽  
Sunhee Yu ◽  
Kyunghee Lee ◽  
Jong-sang Park
Keyword(s):  
Binding Protein ◽  
P53 Gene ◽  
In Vitro Transcription ◽  
The Other ◽  
Rat Tissues ◽  
Transcription Regulator ◽  
Recognition Motif ◽  
Specific Manner ◽  
Recognition Motifs

Two recognition motifs of a 40-kDa NF1-like protein were previously identified in the rat p53 promoter. One is located between -296 and -312 (NF1-like element 1) and the other between -195 and -219 (NF1-like element 2). The latter one was also identified as a NF1/YY1 recognition motif in the human p53 promoter. NF1 or YY1 binds to the motif and regulates the expression of the human p53 gene in a tissue-specific manner. In this study, we investigated the binding protein for NF1-like element 2 in various rat tissues. Unlike the human p53 transcription, an NF1-like protein, not YY1, bound to the motif in every tested tissue: thymus, kidney, and spleen. In vitro transcription assay also confirmed that the NF1-like protein regulated the p53 transcription in rat spleen, although the human p53 transcription was regulated by YY1 in that organ. The molecular mass of the binding protein was determined to be 40 kDa, which was the same as that of the NF1-like protein identified in liver. Therefore, the 40-kDa NF1-like protein may be a universal transcription regulator for the rat p53 gene.Key words: NF1-like protein, p53, promoter, transcription regulation, YY1.

Characteristics of a somatostatin-binding protein

1978 ◽  
Vol 56 (1) ◽  
pp. 48-53 ◽  
Author(s):  
N. Ogawa ◽  
T. Thompson ◽  
H. G. Friesen
Keyword(s):  
Diabetes Mellitus ◽  
Binding Protein ◽  
Gel Filtration ◽  
The Other ◽  
Rat Tissues ◽  
Cytoplasmic Protein ◽  
Disulfide Bridges ◽  
Thiol Groups ◽  
Tissue Extracts ◽  
Streptozotocin Induced Diabetes

The concentrations of a somatostatin-binding protein, found in the cytosol of a number of rat tissues, are similar in both sexes, and hypophysectomy has little or no effect on the level of binding protein in tissue extracts. On the other hand, streptozotocin-induced diabetes mellitus causes a modest decrease. The somatostatin-binding proteins obtained from extracts of several rat tissues are not only similar in molecular weight but also exhibit a similar isoelectric point and electrophoretic mobility. Agents that block thiol groups or prevent the formation of disulfide bridges markedly decrease the binding of somatostatin to the cytoplasmic protein. Studies using thiol reagents and gel filtration suggest that free thiol groups in somatostatin-binding protein are important for the binding of somatostatin.

scholarly journals An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coliSAB11

1998 ◽  
Vol 180 (5) ◽  
pp. 1135-1147 ◽  
Author(s):  
Scott W. Bearden ◽  
Teanna M. Staggs ◽  
Robert D. Perry
Keyword(s):  
Yersinia Pestis ◽  
Cell Envelope ◽  
Binding Protein ◽  
Growth Stimulation ◽  
Iron Chelator ◽  
In Vitro Transcription ◽  
Cosmid Library ◽  
Atp Binding ◽  
Fur Protein

ABSTRACT The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA ofY. pestis KIM6+ was used for transduction of anEscherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribedyfeE gene may be located in the cell envelope, since ayfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

In vitro transcription termination activity of the Drosophila mitochondrial DNA-binding protein DmTTF

2005 ◽  
Vol 331 (1) ◽  
pp. 357-362 ◽  
Author(s):  
Marina Roberti ◽  
Patricio Fernandez-Silva ◽  
Paola Loguercio Polosa ◽  
Erika Fernandez-Vizarra ◽  
Francesco Bruni ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Binding ◽  
Binding Protein ◽  
Transcription Termination ◽  
Dna Binding Protein ◽  
In Vitro Transcription ◽  
Termination Activity

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

Correlation between the activity of a 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi

1988 ◽  
Vol 66 (11) ◽  
pp. 1177-1185 ◽  
Author(s):  
D. Barettino ◽  
G. Morcillo ◽  
J. L. Díez ◽  
M. T. Carretero ◽  
M. J. Carmona
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Specific Inhibitor ◽  
In Vitro Transcription ◽  
The Other ◽  
Low Salt ◽  
Transcription Inhibitor ◽  
Chironomus Thummi

The induction of puff III-A3b, a major heat-shock puff in Chironomus thummi salivary cells, was insensitive to the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), whereas no transcriptional activity could be detected at the other heat-shock puffs in the presence of this drug. In these conditions, a polypeptide with the same Mr and isoform pattern as those of the major heat-shock polypeptide, hsp70, was synthesized. These results suggest that hsp70 is encoded by locus III-A3b. In addition to DRB insensitivity, incorporation of [3H]UTP on puff III-A3b took place in an in vitro transcription assay under low-salt conditions (100 mM NaCl); no labelling could be detected at the other heat-shock puffs under these conditions. Although DRB has been reported as a specific inhibitor of RNA polymerase II-directed transcription, and although the low-salt conditions were not propitious for the activity of this enzyme, RNA polymerase II was detected on puff III-A3b and on the other heat-shock puffs by immunofluorescence with anti-RNA polymerase II antibodies.

scholarly journals Even-skipped Represses Transcription by Binding TATA Binding Protein and Blocking the TFIID-TATA Box Interaction

1998 ◽  
Vol 18 (7) ◽  
pp. 3771-3781 ◽  
Author(s):  
Chi Li ◽  
James L. Manley
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Direct Interaction ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Homeodomain Protein ◽  
Necessary And Sufficient

ABSTRACT The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression. We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.

scholarly journals The Host Factor Polyhedrin Promoter Binding Protein (PPBP) Is Involved in Transcription from the Baculovirus Polyhedrin Gene Promoter

1998 ◽  
Vol 72 (9) ◽  
pp. 7484-7493 ◽  
Author(s):  
Sudip Ghosh ◽  
Anjali Jain ◽  
Bipasha Mukherjee ◽  
Saman Habib ◽  
Seyed E. Hasnain
Keyword(s):  
Binding Protein ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Host Factor ◽  
Luciferase Reporter ◽  
Sequence Motifs ◽  
Luciferase Reporter Gene ◽  
Polyhedrin Promoter

ABSTRACT Hypertranscription and temporal expression from theAutographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an α-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750–2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405–4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda(Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polhpromoter. Replacement of PPBP cognate sequences within thepolh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from thepolh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present intrans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription.

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