scholarly journals Crystal Structure of a Conserved Hypothetical Protein MJ0927 fromMethanocaldococcus jannaschiiReveals a Novel Quaternary Assembly in the Nif3 Family

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Sheng-Chia Chen ◽  
Chi-Hung Huang ◽  
Chia Shin Yang ◽  
Shu-Min Kuan ◽  
Ching-Ting Lin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Hypothetical Protein ◽  
Mobility Shift ◽  
Methanocaldococcus Jannaschii ◽  
Double Stranded Dna ◽  
Family Protein ◽  
Electrophoretic Mobility Shift Assays ◽  
Quaternary Assembly ◽  
Positively Charged

A Nif3 family protein ofMethanocaldococcus jannaschii, MJ0927, is highly conserved from bacteria to humans. Although several structures of bacterial Nif3 proteins are known, no structure representing archaeal Nif3 has yet been reported. The crystal structure ofMethanocaldococcus jannaschiiMJ0927 was determined at 2.47 Å resolution to understand the structural differences between the bacterial and archaeal Nif3 proteins. Intriguingly, MJ0927 is found to adopt an unusual assembly comprising a trimer of dimers that forms a cage-like architecture. Electrophoretic mobility-shift assays indicate that MJ0927 binds to both single-stranded and double-stranded DNA. Structural analysis of MJ0927 reveals a positively charged region that can potentially explain its DNA-binding capability. Taken together, these data suggest that MJ0927 adopts a novel quartenary architecture that could play various DNA-binding roles inMethanocaldococcus jannaschii.

The crystal structure of the Sox4 HMG domain–DNA complex suggests a mechanism for positional interdependence in DNA recognition

2012 ◽  
Vol 443 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Ralf Jauch ◽  
Calista K. L. Ng ◽  
Kamesh Narasimhan ◽  
Prasanna R. Kolatkar
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Dna Recognition ◽  
High Mobility ◽  
Mobility Shift ◽  
Binding Motifs ◽  
Binding Interface ◽  
Structural Mechanisms ◽  
Electrophoretic Mobility Shift Assays ◽  
Dna Complex

It has recently been proposed that the sequence preferences of DNA-binding TFs (transcription factors) can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. In the present study we examine the crystal structure of the HMG (high-mobility group) domain of Sox4 [Sry (sex-determining region on the Y chromosome)-related HMG box 4] bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2, we observed subtle conformational differences at the DNA-binding interface. Furthermore, using quantitative electrophoretic mobility-shift assays we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These results suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adaptations lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.

scholarly journals Characterization of the Single-Stranded DNA Binding Protein Encoded by the Vaccinia Virus I3 Gene

1998 ◽  
Vol 72 (4) ◽  
pp. 2917-2926 ◽  
Author(s):  
S. Craig Rochester ◽  
Paula Traktman
Keyword(s):  
Dna Binding ◽  
Vaccinia Virus ◽  
Dna Binding Protein ◽  
Mobility Shift ◽  
Single Stranded Dna ◽  
Charged Amino Acids ◽  
Double Stranded Dna ◽  
Infected Cells ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed inEscherichia coli and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.

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.

scholarly journals Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake

2020 ◽  
Vol 6 (35) ◽  
pp. eaba7637
Author(s):  
Yoshiki Tanaka ◽  
Kunihito Yoshikaie ◽  
Azusa Takeuchi ◽  
Muneyoshi Ichikawa ◽  
Tomoyuki Mori ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sulfur Cycle ◽  
Sulfur Source ◽  
Bacterial Membrane ◽  
Density Maps ◽  
Family Protein ◽  
Cysteine Synthesis ◽  
Plausible Mechanism ◽  
Dynamics Simulations ◽  
Positively Charged

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

Crystal structure analysis of a hypothetical protein (MJ0366) from Methanocaldococcus jannaschii revealed a novel topological arrangement of the knot fold

2017 ◽  
Vol 482 (2) ◽  
pp. 264-269 ◽  
Author(s):  
Viswanathan Thiruselvam ◽  
Thirumananseri Kumarevel ◽  
Ponnuraj Karthe ◽  
Seiki Kuramitsu ◽  
Shigeyuki Yokoyama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structure Analysis ◽  
Hypothetical Protein ◽  
Crystal Structure Analysis ◽  
Methanocaldococcus Jannaschii

scholarly journals Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2)

2014 ◽  
Vol 42 (15) ◽  
pp. 10122-10133 ◽  
Author(s):  
Hussain Bhukya ◽  
Ruchika Bhujbalrao ◽  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Dna Sequences ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Antibiotic Resistant ◽  
Transporter Protein ◽  
Dimeric Interface ◽  
Transcriptional Regulatory ◽  
Family Protein

AbstractAntibiotic production and resistance pathways in Streptomyces are dictated by the interplay of transcriptional regulatory proteins that trigger downstream responses via binding to small diffusible molecules. To decipher the mode of DNA binding and the associated allosteric mechanism in the sub-class of transcription factors that are induced by γ-butyrolactones, we present the crystal structure of CprB in complex with the consensus DNA element to a resolution of 3.25 Å. Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove. The crystal structure revealed that, CprB is bound to DNA as a dimer of dimers with the mode of binding being analogous to the broad spectrum multidrug transporter protein QacR from the antibiotic resistant strain Staphylococcus aureus. It was demonstrated that the CprB displays a cooperative mode of DNA binding, following a clamp and click model. Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator. Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).

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 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.

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