β-Arm flexibility of HU fromStaphylococcus aureusdictates the DNA-binding and recognition mechanism

2014 ◽  
Vol 70 (12) ◽  
pp. 3273-3289 ◽  
Author(s):  
Do-Hee Kim ◽  
Hookang Im ◽  
Jun-Goo Jee ◽  
Sun-Bok Jang ◽  
Hye-Jin Yoon ◽  
...  
Keyword(s):  
Dna Binding ◽  
Md Simulations ◽  
Titration Calorimetry ◽  
Salt Bridges ◽  
Mobility Shift ◽  
Bent Dna ◽  
Recognition Mechanism ◽  
Local Shape ◽  
Associated Proteins ◽  
Electrophoretic Mobility Shift Assays

HU, one of the major nucleoid-associated proteins, interacts with the minor groove of DNA in a nonspecific manner to induce DNA bending or to stabilize bent DNA. In this study, crystal structures are reported for both free HU fromStaphylococcus aureusMu50 (SHU) and SHU bound to 21-mer dsDNA. The structures, in combination with electrophoretic mobility shift assays (EMSAs), isothermal titration calorimetry (ITC) measurements and molecular-dynamics (MD) simulations, elucidate the overall and residue-specific changes in SHU upon recognizing and binding to DNA. Firstly, structural comparison showed the flexible nature of the β-sheets of the DNA-binding domain and that the β-arms bend inwards upon complex formation, whereas the other portions are nearly unaltered. Secondly, it was found that the disruption and formation of salt bridges accompanies DNA binding. Thirdly, residue-specific free-energy analyses using the MM-PBSA method with MD simulation data suggested that the successive basic residues in the β-arms play a central role in recognizing and binding to DNA, which was confirmed by the EMSA and ITC analyses. Moreover, residue Arg55 resides in the hinge region of the flexible β-arms, exhibiting a remarkable role in their flexible nature. Fourthly, EMSAs with various DNAs revealed that SHU prefers deformable DNA. Taken together, these data suggest residue-specific roles in local shape and base readouts, which are primarily mediated by the flexible β-arms consisting of residues 50–80.

Constitutive activation of LIL-Stat in adult T-cell leukemia cells

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2715-2718 ◽  
Author(s):  
Junichi Tsukada ◽  
Yoko Toda ◽  
Masahiro Misago ◽  
Yoshiya Tanaka ◽  
Philip E. Auron ◽  
...  
Keyword(s):  
T Cell ◽  
Dna Binding ◽  
Leukemic Cells ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Mobility Shift ◽  
Adult T Cell Leukemia ◽  
Nuclear Extracts ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1–inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1–responsive element), which is found in the human prointerleukin 1β (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL-1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in theIL1B gene.

RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication

1990 ◽  
Vol 10 (12) ◽  
pp. 6236-6243
Author(s):  
M S Caddle ◽  
L Dailey ◽  
N H Heintz
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Bending ◽  
Chinese Hamster ◽  
Nuclear Extract ◽  
Dna Fragments ◽  
Mobility Shift ◽  
Bent Dna ◽  
Origin Region ◽  
Gel Mobility Shift

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.

Constitutive activation of LIL-Stat in adult T-cell leukemia cells

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2715-2718
Author(s):  
Junichi Tsukada ◽  
Yoko Toda ◽  
Masahiro Misago ◽  
Yoshiya Tanaka ◽  
Philip E. Auron ◽  
...  
Keyword(s):  
T Cell ◽  
Dna Binding ◽  
Leukemic Cells ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Mobility Shift ◽  
Adult T Cell Leukemia ◽  
Nuclear Extracts ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1–inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1–responsive element), which is found in the human prointerleukin 1β (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL-1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in theIL1B gene.

scholarly journals Protein-Protein Interaction between Fli-1 and GATA-1 Mediates Synergistic Expression of Megakaryocyte-Specific Genes through Cooperative DNA Binding

2003 ◽  
Vol 23 (10) ◽  
pp. 3427-3441 ◽  
Author(s):  
Michael Eisbacher ◽  
Melissa L. Holmes ◽  
Anthea Newton ◽  
Philip J. Hogg ◽  
Levon M. Khachigian ◽  
...  
Keyword(s):  
Dna Binding ◽  
K562 Cells ◽  
Physical Interaction ◽  
Mobility Shift ◽  
Megakaryocytic Differentiation ◽  
Protein Protein Interaction ◽  
Expression Of Genes ◽  
Friend Leukemia ◽  
Ets Domain ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbα in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbα genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.

scholarly journals RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

1990 ◽  
Vol 10 (12) ◽  
pp. 6236-6243 ◽  
Author(s):  
M S Caddle ◽  
L Dailey ◽  
N H Heintz
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Bending ◽  
Chinese Hamster ◽  
Nuclear Extract ◽  
Dna Fragments ◽  
Mobility Shift ◽  
Bent Dna ◽  
Origin Region ◽  
Gel Mobility Shift

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.

scholarly journals Dynamical Behavior of the Human Ferroportin Homologue from Bdellovibrio bacteriovorus: Insight into the Ligand Recognition Mechanism

2020 ◽  
Vol 21 (18) ◽  
pp. 6785
Author(s):  
Valentina Tortosa ◽  
Maria Carmela Bonaccorsi di Patti ◽  
Federico Iacovelli ◽  
Andrea Pasquadibisceglie ◽  
Mattia Falconi ◽  
...  
Keyword(s):  
Dynamical Behavior ◽  
Md Simulations ◽  
Major Facilitator Superfamily ◽  
Ligand Recognition ◽  
Salt Bridges ◽  
Recognition Mechanism ◽  
Bdellovibrio Bacteriovorus ◽  
Physiological Relevance ◽  
Bacteria And Fungi ◽  
Major Facilitator

Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer’s disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein’s dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.

scholarly journals The Sequence-specific DNA Binding of NF-κB Is Reversibly Regulated by the Automodification Reaction of Poly (ADP-ribose) Polymerase 1

2001 ◽  
Vol 276 (50) ◽  
pp. 47664-47670 ◽  
Author(s):  
Woo-Jin Chang ◽  
Rafael Alvarez-Gonzalez
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Specific Binding ◽  
High Specificity ◽  
Immunoblot Analysis ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays ◽  
Parp 1

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+in vitro.Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activationin vivoas well.

scholarly journals Modulation of interferon signaling in human fibroblasts by phorbol esters.

1992 ◽  
Vol 12 (10) ◽  
pp. 4486-4495 ◽  
Author(s):  
E F Petricoin ◽  
R H Hackett ◽  
H Akai ◽  
K Igarashi ◽  
D S Finbloom ◽  
...  
Keyword(s):  
Dna Binding ◽  
Electrophoretic Mobility ◽  
Transcription Initiation ◽  
Phorbol Esters ◽  
Human Fibroblasts ◽  
Substantial Reduction ◽  
Cellular Gene ◽  
Mobility Shift ◽  
Ifn Alpha ◽  
Electrophoretic Mobility Shift Assays

Phorbol esters activate the expression of a variety of early-response genes through protein kinase C-dependent pathways. In addition, phorbol esters may promote cell growth by the inhibition of expression of cellular gene products regulated by antiproliferative agents such as interferons (IFN)s. In human diploid fibroblasts, phorbol 12-myristate 13-acetate (PMA) selectively inhibits the IFN-alpha-induced cellular gene ISG54. Using transient transfection assays, we have delineated two elements in the promoter of this gene that are necessary for the inhibitory actions of PMA. These elements include (i) the IFN-stimulated response element (ISRE) which is necessary for IFN-alpha-induced cellular gene expression, and (ii) an element located near the site of transcription initiation. IFN-alpha treatment resulted in the rapid induction of ISGF3, a multisubunit transcription factor which binds to the ISRE. PMA caused a substantial reduction in IFN alpha-induced ISGF3 in both nuclear and cytoplasmic extracts, as determined by electrophoretic mobility shift assays with the ISRE as a probe. In vitro reconstitution experiments revealed that IFN-alpha activation of the ISGF3 alpha component of ISGF3 was not affected by PMA. Further experiments were consistent with the possibility that PMA regulated the activity of a cellular factor which competed with ISGF3 gamma for binding of the activated ISGF3 alpha polypeptides. Electrophoretic mobility shift assays using the cap site of ISG54 as a probe demonstrated the formation of a specific complex whose DNA binding activity was not affected by treatment of cells with PMA or IFN-alpha. Competitive inhibition studies were consistent with the DNA-protein complex at the cap site of ISG54 containing proteins with DNA binding sites in common with those which also interact with the ISRE. These data suggest a unique regulatory mechanism by which phorbol esters can modulate IFN signaling.

scholarly journals P64, a Novel Major Virion DNA-Binding Protein Potentially Involved in Condensing the Spodoptera frugiperda Ascovirus 1a Genome

2009 ◽  
Vol 83 (6) ◽  
pp. 2708-2714 ◽  
Author(s):  
Yeping Tan ◽  
Tatsinda Spears ◽  
Dennis K. Bideshi ◽  
Jeffrey J. Johnson ◽  
Robert Hice ◽  
...  
Keyword(s):  
Dna Binding ◽  
Spodoptera Frugiperda ◽  
Consensus Sequence ◽  
Structural Protein ◽  
Mobility Shift ◽  
Virion Assembly ◽  
Amino Terminal ◽  
Ultrathin Sections ◽  
Carboxy Terminal ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT We recently identified 21 structural proteins in the virion of Spodoptera frugiperda ascovirus 1a (SfAV1a), a virus with a large, double-stranded DNA genome of 157 kbp, which attacks species of the lepidopteran family Noctuidae. The two most abundant virion proteins were the major capsid protein and a novel protein (P64) of 64 kDa that contained two distinct domains not known previously to occur together. The amino-terminal half of P64 (residues 1 to 263) contained four repeats (a recently recognized motif with an unknown function) of a virus-specific two-cysteine adaptor. Adjoined to this, the carboxy-terminal half of P64 (residues 279 to 455) contained 14 copies of a highly basic, tandemly repeated motif rich in arginine and serine, having an 11- to 13-amino-acid consensus sequence, SPSQRRSTS(V/K)(A/S)RR, yielding a predicted isoelectric point of 12.2 for this protein. In the present study, we demonstrate by Southwestern analysis that SfAV1a P64 was the only virion structural protein that bound DNA. Additional electrophoretic mobility shift assays showed that P64 bound SfAV1a as well as non-SfAV1a DNA. Furthermore, we show through immunogold labeling of ultrathin sections that P64 is a component of virogenic stroma and appears to be progressively incorporated into the SfAV1a DNA core during virion assembly. As no other virion structural protein bound DNA and no basic DNA-binding proteins of lower mass are encoded by the SfAV1a genome or were identified by proteomic analysis, our results suggest that P64's function is to condense the large genome of this virus and assist in packaging this genome into its virion.

scholarly journals DIMR, a Yeast-Based Synthetic Reporter System for Probing Oligomeric Transcription Factor DNA Binding

2019 ◽  
Author(s):  
Zachary A. Myers ◽  
Swadhin Swain ◽  
Shannan Bialek ◽  
Samuel Keltner ◽  
Ben F. Holt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Reporter System ◽  
Mobility Shift ◽  
Genomic Locus ◽  
Biological Regulation ◽  
Transcriptional Regulatory ◽  
Regulatory Capacity ◽  
Electrophoretic Mobility Shift Assays ◽  
Y Family

AbstractTranscription factors (TFs) are fundamental components of biological regulation, facilitating the basal and differential gene expression necessary for life. TFs exert transcriptional regulation through interactions with both DNA and other TFs, ultimately influencing the action of RNA polymerase at a genomic locus. Current approaches are proficient at identification of binding site requirements for individual TFs, but few methods have been adapted to study oligomeric TF complexes. Further, many approaches that have been turned toward understanding DNA binding of TF complexes, such as electrophoretic mobility shift assays, require protein purification steps that can be burdensome or scope-limiting when considering more exhaustive experimental design. In order to address these shortfalls and to facilitate a more streamlined approach to understanding DNA binding by TF complexes, we developed the DIMR (Dynamic, Interdependent TF binding Molecular Reporter) system, a modular, yeast-based synthetic transcriptional activity reporter. As a proof of concept, we focused on the NUCLEAR FACTOR-Y (NF-Y) family of obligate heterotrimeric TFs in Arabidopsis thaliana. The DIMR system was able to reproduce the strict DNA-binding requirements of an experimentally validated NF-YA2/B2/C3 complex with high fidelity, including recapitulation of previously characterized mutations in subunits that either break NF-Y complex interactions or are directly involved in DNA binding. The DIMR system is a novel, powerful, and easy-to-use approach to address questions regarding the binding of oligomeric TFs to DNA.One sentence summaryThe DIMR system provides an accessible and easy-to-use platform to elucidate DNA binding and transcriptional regulatory capacity of oligomeric transcription factor complexes

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