Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs

1993 ◽  
Vol 13 (7) ◽  
pp. 4167-4173
Author(s):  
J Min ◽  
H P Zassenhaus
Keyword(s):  
Binding Activity ◽  
Nucleoside Triphosphate ◽  
Yeast Mitochondria ◽  
Mobility Shift ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Molecular Masses ◽  
Labeled Rna ◽  
Gel Mobility Shift ◽  
A Site

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.

scholarly journals Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs.

1993 ◽  
Vol 13 (7) ◽  
pp. 4167-4173 ◽  
Author(s):  
J Min ◽  
H P Zassenhaus
Keyword(s):  
Binding Activity ◽  
Nucleoside Triphosphate ◽  
Yeast Mitochondria ◽  
Mobility Shift ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Molecular Masses ◽  
Labeled Rna ◽  
Gel Mobility Shift ◽  
A Site

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.

DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 281-290 ◽  
Author(s):  
Yu. Rebets ◽  
B. Ostash ◽  
A. Luzhetskyy ◽  
S. Kushnir ◽  
M. Fukuhara ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Gene Promoter ◽  
Binding Activity ◽  
Mobility Shift ◽  
Chitin Binding Domain ◽  
Dna Binding Activity ◽  
Gel Mobility Shift

The gene lndI is involved in the pathway-specific positive regulation of biosynthesis of the antitumour polyketide landomycin E in Streptomyces globisporus 1912. LndI was overexpressed in Escherichia coli as a protein C-terminally fused to the intein-chitin-binding-domain tag and purified in a one-step column procedure. Results of in vivo LndI titration, DNA gel mobility-shift assays and promoter-probing experiments indicate that LndI is an autoregulatory DNA-binding protein that binds to its own gene promoter and to the promoter of the structural gene lndE. Enhanced green fluorescent protein was used as a reporter to study the temporal and spatial pattern of lndI transcription. Expression of lndI started before cells entered mid-exponential phase and peak expression coincided with maximal accumulation of landomycin E and biomass. In solid-phase analysis, lndI expression was evident in substrate mycelia but was absent from aerial hyphae and spores.

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.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1065-C1072 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Inos Gene ◽  
E Box ◽  
Inos Promoter ◽  
Gel Mobility Shift

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.

scholarly journals Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region

2005 ◽  
Vol 25 (10) ◽  
pp. 3855-3863 ◽  
Author(s):  
David J. Katz ◽  
Michael A. Beer ◽  
John M. Levorse ◽  
Shirley M. Tilghman
Keyword(s):  
Binding Sites ◽  
Zinc Finger Protein ◽  
Functional Characterization ◽  
Binding Activity ◽  
Mobility Shift ◽  
Control Center ◽  
Dna Binding Activity ◽  
A Genome ◽  
Pause Site

ABSTRACT The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg2+, implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

scholarly journals The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro.

1997 ◽  
Vol 17 (10) ◽  
pp. 5923-5934 ◽  
Author(s):  
H Lu ◽  
R P Fisher ◽  
P Bailey ◽  
A J Levine
Keyword(s):  
Transcription Factor ◽  
Excision Repair ◽  
Binding Activity ◽  
Mobility Shift ◽  
Trimeric Complex ◽  
Dna Binding Activity ◽  
Terminal Amino ◽  
Gel Mobility Shift ◽  
Kinase Phosphorylation

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.

IL-17 stimulates inflammatory responses via NF-κB and MAP kinase pathways in human colonic myofibroblasts

2002 ◽  
Vol 282 (6) ◽  
pp. G1035-G1044 ◽  
Author(s):  
Kazunori Hata ◽  
Akira Andoh ◽  
Mitsue Shimada ◽  
Sanae Fujino ◽  
Shigeki Bamba ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Northern Blotting ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Tnf Α ◽  
Mobility Shift Assay ◽  
Mapk Inhibitors ◽  
Gel Mobility Shift

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-κB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-κB activation within 45 min after stimulation. A blockade of NF-κB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1β or IL-17 + tumor necrosis factor (TNF)-α enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-α on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.

scholarly journals Characterization of a telomere-binding protein from Physarum polycephalum.

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290 ◽  
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

scholarly journals PDGF β-Receptor Kinase Activity and ERK1/2 Mediate Glycosaminoglycan Elongation on Biglycan and Increases Binding to LDL

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4356-4367 ◽  
Author(s):  
Robel Getachew ◽  
Mandy L. Ballinger ◽  
Micah L. Burch ◽  
Julianne J. Reid ◽  
Levon M. Khachigian ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Structural Characteristics ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Mobility Shift ◽  
Downstream Signaling ◽  
Β Receptor ◽  
Gel Mobility Shift

The initiation of atherosclerosis involves the subendothelial retention of lipoproteins by proteoglycans (PGs). Structural characteristics of glycosaminoglycan (GAG) chains on PGs influence lipoprotein binding and are altered adversely by platelet-derived growth factor (PDGF). The signaling pathway for PDGF-mediated GAG elongation via the PDGF receptor (PDGFR) was investigated. In human vascular smooth muscle cells, PDGF significantly increased 35S-sulfate incorporation into PGs and GAG chain size. PGs from PDGF-stimulated cells showed increased binding low-density lipoprotein (P < 0.001) in gel mobility shift assays. Knockdown of PDGFRβ using small interfering RNA demonstrated that PDGF mediated changes in PGs via PDGFRβ. GAG synthesis and hyperelongation was blocked by inhibition of receptor tyrosine kinase autophosphorylation site Tyr857 activity using Ki11502 or imatinib. Downstream signaling to GAG hyperelongation was mediated through ERK MAPK and not phosphatidylinositol-3 kinase or phospholipase Cγ. In high-fat-fed apolipoprotein E−/− mice, inhibition of PDGFRβ activity by imatinib reduced aortic total lipid staining area by 35% (P < 0.05). Inhibition of PDGFRβ tyrosine kinase activity leads to inhibition of GAG synthesis on vascular PGs and aortic lipid area in vivo. PDGFRβ and its signaling pathways are potential targets for novel therapeutic agents to prevent the earliest stages atherosclerosis.

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