A downstream sequence of the rpL32 promoter competes with the glucocorticoid responsive element for a protein factor

1992 ◽  
Vol 70 (9) ◽  
pp. 787-791 ◽  
Author(s):  
Thillainathan Yoganathan ◽  
Bruce H. Sells
Keyword(s):  
Ribosomal Protein ◽  
Cross Linking ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
Protein Factor ◽  
In The Wild ◽  
Glucocorticoid Responsive Element ◽  
Mobility Shift Assay ◽  
Responsive Element ◽  
Gel Mobility Shift

The murine ribosomal protein (rp) L32 gene contains essential promoter sequences located both upstream and downstream of the cap site. A combination of gel mobility shift, UV cross-linking, and cell-free transcription assays were used to analyze the interaction of factors binding to a downstream element (located at position +25 to +37). The rpL32 downstream element identified polypeptides (transcription factors) ranging in size from 45 to 25 kilodaltons (kDa). Four base pair changes in the wild-type sequence of the downstream element eliminated binding. An oligonucleotide containing the glucocorticoid responsive element sequence competed specifically for the 45-kDa protein in both the gel mobility shift assay and in the UV cross-linking studies. Our data also indicate that the downstream binding factors contribute to cell-free transcription of the rpL32 gene.Key words: ribosomal protein L32, transcription factor, glucocorticoid response element.

scholarly journals Pyrazole-inducible proteins in DBA/2 mouse liver bind with high affinity to the 3′-untranslated regions of the mRNAs of coumarin hydroxylase (CYP2A5) and c-jun

1998 ◽  
Vol 331 (2) ◽  
pp. 473-481 ◽  
Author(s):  
Monika THULKE-GROSS ◽  
Manfred HERGENHAHN ◽  
Anne TILLOY-ELLUL ◽  
Matti LANG ◽  
Helmut BARTSCH
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Binding Activity ◽  
Cross Linking ◽  
Mobility Shift ◽  
Gel Mobility Shift Assay ◽  
Molecular Masses ◽  
Fold Induction ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

An important mechanism in the up-regulation of cytochrome P-450 2A5 (CYP2A5, coumarin hydroxylase, Coh) is the stabilization of the corresponding mRNA; some evidence suggests that proteins binding to CYP2A5 mRNA may be involved in this stabilization. Here we report that pyrazole, a well known inducer of CYP2A5 and stabilizer of its message, enhances the binding of a set of proteins to 32P-labelled 3´-untranslated region (3´UTR) of CYP2A5 to give 32P-labelled bands of apparent molecular mass 37/39, 45/48 and 70/72 kDa after UV cross-linking/RNase cleavage; in addition, we found different proteins binding to other parts of CYP2A5 mRNA. The 70/72 kDa bands are also formed with the 3´UTR of c-jun. The inducible proteins are found in different cellular subfractions at different concentrations, with a maximum of five-fold induction of binding activity in microsomes. When a gel-mobility-shift assay was combined with UV cross-linking to resolve different pyrazole-inducible RNA–protein complexes into single RNA-binding protein bands, the smallest complex contained a double band of 37/39 kDa, 45/48 kDa bands, 70/72 kDa bands, and additional weaker bands at higher molecular masses (around 120 kDa). This composition was found also for all other complexes detected by gel-mobility-shift assay; occasionally, bands at higher molecular masses were also observed. The proteins of the smallest complex might therefore represent a core with which other proteins interact to build up larger complexes. Binding of proteins 37/39 kDa and 70/72 kDa was located to a 20-base loop and adjacent sequences in a 70 nt AU-rich region of the 3´UTR of the CYP2A5. Based on our previous evidence, this 70-nt sequence may play an important role in the stabilization and processing of the message.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals Identification of the proteins responsible for SAR DNA binding in nuclear matrix of Cucurbita pepo.

1995 ◽  
Vol 42 (2) ◽  
pp. 171-176
Author(s):  
R Rzepecki ◽  
E Markiewicz ◽  
J Szopa
Keyword(s):  
Dna Binding ◽  
Cucurbita Pepo ◽  
Standard Procedure ◽  
Cell Nuclei ◽  
Mobility Shift ◽  
Gel Mobility Shift Assay ◽  
Mobility Shift Assay ◽  
Pre Treatment ◽  
Gel Mobility Shift ◽  
Triton X

The nuclear matrices from White bush (Cucurbita pepo var. patisonina) cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human beta-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments.

A Gel Mobility Shift Assay for Probing the Effect of Drug–DNA Adducts on DNA-Binding Proteins

2003 ◽  
pp. 95-106 ◽  
Author(s):  
Suzanne M. Cutts ◽  
Andrew Masta ◽  
Con Panousis ◽  
Peter G. Parsons ◽  
Richard A. Sturm ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Adducts ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Mobility Shift ◽  
Gel Mobility Shift Assay ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

scholarly journals Escherichia coli FadR Positively Regulates Transcription of the fabB Fatty Acid Biosynthetic Gene

2001 ◽  
Vol 183 (20) ◽  
pp. 5982-5990 ◽  
Author(s):  
John W. Campbell ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Protein A ◽  
Transcriptional Level ◽  
Biosynthetic Gene ◽  
Biochemical Tests ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

ABSTRACT In Escherichia coli expression of the genes of fatty acid degradation (fad) is negatively regulated at the transcriptional level by FadR protein. In contrast the unsaturated fatty acid biosynthetic gene, fabA, is positively regulated by FadR. We report that fabB, a second unsaturated fatty acid biosynthetic gene, is also positively regulated by FadR. Genomic array studies that compared global transcriptional differences between wild-type and fadR-null mutant strains, as well as in cultures of each strain grown in the presence of exogenous oleic acid, indicated that expression of fabBwas regulated in a manner very similar to that of fabAexpression. A series of genetic and biochemical tests confirmed these observations. Strains containing both fabB andfadR mutant alleles were constructed and shown to exhibit synthetic lethal phenotypes, similar to those observed infabA fadR mutants. A fadR strain was hypersensitive to cerulenin, an antibiotic that at low concentrations specifically targets the FabB protein. A transcriptional fusion of chloramphenicol acetyltransferase (CAT) to the fabBpromoter produces lower levels of CAT protein in a strain lacking functional FadR. The ability of a putative FadR binding site within thefabB promoter to form a complex with purified FadR protein was determined by a gel mobility shift assay. These experiments demonstrate that expression of fabB is positively regulated by FadR.

scholarly journals Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

1992 ◽  
Vol 12 (1) ◽  
pp. 413-421 ◽  
Author(s):  
P Cortes ◽  
O Flores ◽  
D Reinberg
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Stable Complex ◽  
Mobility Shift ◽  
Yeast Saccharomyces Cerevisiae ◽  
Gel Mobility Shift Assay ◽  
Two Factors ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Tfiid Complex

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

scholarly journals Control of Enzyme IIscr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon

2003 ◽  
Vol 185 (19) ◽  
pp. 5791-5799 ◽  
Author(s):  
Bing Wang ◽  
Howard K. Kuramitsu
Keyword(s):  
Streptococcus Mutans ◽  
Transcriptional Repressor ◽  
Direct Repeat ◽  
Dnase I ◽  
Repeat Sequence ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
In The Wild ◽  
Dna Binding Properties

ABSTRACT In Streptococcus mutans, enzyme IIscr and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme IIscr and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted in increased expression of scrB relative to that in the wild-type strain. In this study, we employed DNA mobility shift and DNase I protection assays with a purified ScrR-histidine tag fusion protein to examine the DNA binding properties of ScrR to the promoter regions of the scrA and scrB genes. The results showed that ScrR bound specifically to the promoter regions of both scrA and scrB. Two regions with high affinity for ScrR in the promoter sequences of the scrA and scrB genes were identified by DNase I protection assays. One, O C, which includes a 20-bp imperfect inverted-repeat sequence, is located between the two promoters, and the other, O B, is located within the scrB promoter region containing a 37-bp imperfect direct-repeat sequence. Mutations of O B and O C resulted in constitutive transcription and expression of both the scrA and scrB genes. Our results indicated that S. mutans coordinates the activities of enzyme IIscr and sucrose-6-phosphate hydrolase by transcriptional repressor ScrR binding to the promoter regions of the scr regulon.

scholarly journals Detection of Proteins that Recognize Platinum-modified DNA Using Gel Mobility Shift Assay

1990 ◽  
Vol 81 (12) ◽  
pp. 1210-1213 ◽  
Author(s):  
Yasuhiro Fujiwara ◽  
Kazuo Kasahara ◽  
Yoshikazu Sugimoto ◽  
Kazuto Nishio ◽  
Tohru Ohmori ◽  
...  
Keyword(s):  
Mobility Shift ◽  
Gel Mobility Shift Assay ◽  
Modified Dna ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

scholarly journals Regulation of Expression of theyiaKLMNOPQRS Operon for Carbohydrate Utilization inEscherichia coli: Involvement of the Main Transcriptional Factors

2000 ◽  
Vol 182 (16) ◽  
pp. 4617-4624 ◽  
Author(s):  
Ester Ibañez ◽  
Evangelina Campos ◽  
Laura Baldoma ◽  
Juan Aguilar ◽  
Josefa Badia
Keyword(s):  
Mutant Strain ◽  
Integration Host Factor ◽  
Anaerobic Growth ◽  
Receptor Protein ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Carbohydrate Utilization ◽  
In The Wild ◽  
Gel Mobility Shift ◽  
Transcriptional Fusions

ABSTRACT The yiaKLMNOPQRS (yiaK-S) gene cluster ofEscherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate l-xylulose. Transcription ofyiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study. The 5′ end has been located at 60 bp upstream from the ATG. Expression of theyiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ. No inducer molecule of theyiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. ThelacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from −50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. ThelacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at −106, is essential for yiaK-S expression. The 5′ end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-Spromoter. Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of theyiaK-S promoter.

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