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 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.

scholarly journals TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution

2005 ◽  
Vol 387 (2) ◽  
pp. 401-409 ◽  
Author(s):  
Jolanta KOPEC ◽  
Alexander BERGMANN ◽  
Gerhard FRITZ ◽  
Elisabeth GROHMANN ◽  
Walter KELLER
Keyword(s):  
Amino Acids ◽  
Broad Host Range ◽  
Mobility Shift ◽  
Gram Positive ◽  
Nick Site ◽  
Α Helix ◽  
Electrophoretic Mobility Shift Assays ◽  
Dna Complex ◽  
Β Sheet

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase–oriT complex.

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.

Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A

Science ◽  
1991 ◽  
Vol 252 (5007) ◽  
pp. 809-817 ◽  
Author(s):  
N. Pavletich ◽  
C. Pabo
Keyword(s):  
Crystal Structure ◽  
Zinc Finger ◽  
Dna Recognition ◽  
Dna Complex

Intracellular HMGB1 Transactivates the Human IL1B Gene Promoter through Association with an ETS Transcription Factor PU.1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1271-1271
Author(s):  
Junichi Tsukada ◽  
Fumihiko Mouri ◽  
Takamitsu Mizobe ◽  
Takehiro Higashi ◽  
Hiroto Izumi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Proinflammatory Cytokine ◽  
Expression Vector ◽  
High Mobility ◽  
Gene Promoter ◽  
Physical Interaction ◽  
Chromosomal Protein ◽  
Mobility Shift

Abstract High mobility group box chromosomal protein 1 (HMGB1) was recently identified as a late proinflammatory mediator of endotoxin lethality, which amplifies and sustains the inflammatory processes via macrophage/monocyte activation by extracellular release of HMGB1. However, it was originally described as a nonhistone nuclear protein to bind and distort the structure of DNA and to regulate transcription through association with several transcription factors. Here we investigated the effect of intracellular HMGB1 on transcriptional gene regulation of an immediate early proinflammatory cytokine IL-1β in transient transfection studies using HMGB1 expression vector (pcDNA3HMGB1) and murine macrophage cells RAW264.7. When pcDNA3HMGB1 was introduced into RAW264.7 cells with pGL3HT reporter containing the -131 to +12 minimal IL-1β gene promoter, IL-1β promoter activity was significantly induced by expression of HMGB1. The gene promoter possesses two important transcription factor binding motifs, one for PU.1, a myeloid and B cell-specific transcription factor that belongs to the ETS family, and the other a binding site for NF-IL6. To verify the functional role of intracellular HMGB1 in transactivation of the IL-1β promoter, a PU.1 expression vector and/or pcDNA3HMGB1 were cotransfected into PU.1-deficient murine thymocytes EL4 cells along with pGL3HT reporter. As a result, HMGB1 synergized with PU.1 to transactivate the IL-1β promoter, but not HMGB1 alone. This argument was supported by our GST-pulldown data, which demonstrated direct physical interaction of HMGB1 with PU.1. In addition, deletion of the PU.1 winged helix-turn-helix DNA-binding domain significantly inhibited the association of PU.1 with HMGB1. To determine whether HMGB1 could affect PU.1 DNA-binding affinity, we performed electrophoretic mobility shift assay using a radiolabeled IL-1β -59 to +12 promoter element (DT), recombinant PU.1 (rPU.1) and GST-HMGB1. Two complexes with slower and faster mobilities were generated by the addition of HMGB1 to a mixture of rPU.1 and DT probe. The two complexes were abrogated by preincubation with anti-PU.1 Ab, while anti-HMGB1 Ab reacted only with the complex with a slower mobility, indicating that the complex with a slower mobility formed by addition of HMGB1 contained both HMGB1 and PU.1, while the band with a faster mobility contained only PU.1. From the present study, we propose that intracellular HMGB1 might function as a coactivator in PU.1-mediated transcriptional activation, which facilitate access of PU.1 to specific DNA targets. The fact that PU.1 is a transcription factor essential to macrophages/monocyte-specific proinflammatory cytokine genes further raise the possibility that interaction of PU.1 with HMGB1 may play an important role in the inflammation cascade in macrophages/monocytes.

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.

Sign in / Sign up

Export Citation Format

Share Document