scholarly journals Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription

2014 ◽  
Vol 34 (15) ◽  
pp. 2929-2943 ◽  
Author(s):  
I. Kamenova ◽  
L. Warfield ◽  
S. Hahn
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Binding Surface

scholarly journals Crystal structure of chromo barrel domain of RBBP1

2018 ◽  
Author(s):  
Ming Lei ◽  
Yue Feng ◽  
Mengqi Zhou ◽  
Yuan Yang ◽  
Peter Loppnau ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Dna Binding ◽  
Surface Charge ◽  
Gene Transcription ◽  
Molecular Basis ◽  
Binding Ability ◽  
Histone Binding ◽  
Multidomain Protein ◽  
Binding Surface

AbstractRBBP1 is a retinoblastoma protein (pRb) binding protein acting as a repressor of gene transcription. RBBP1 is a multidomain protein including a chromo barrel domain, and its chromo barrel domain has been reported to recognize histone H4K20me3 weakly, and this binding is enhanced by the simultaneous binding of DNA. However, the molecular basis of this DNA-mediated histone binding by the chromo barrel domain of RBBP1 is unclear. Here we attempted to co-crystallize the chromo barrel domain of RBBP1 with either a histone H4K20me3 peptide alone or with both a histone H4K20me3 peptide and DNA, but only solved the peptide/DNA unbound crystal structure. Our structural analysis indicates that RBBP1 could interact with histone H4K20me3 similar to other histone binding chromo barrel domains, and the surface charge representation analysis of the chromo barrel domain of RBBP1 suggests that the chromo barrel domain of RBBP1 does not have a typical DNA binding surface, indicating that it might not bind to DNA. Consistently, our ITC assays also showed that DNA does not significantly enhance the histone binding ability of the chromo barrel domain of RBBP1.

A direct requirement of nuclear factor-κB for suppression of apoptosis in ventricular myocytes

2000 ◽  
Vol 279 (3) ◽  
pp. H939-H945 ◽  
Author(s):  
Shareef Mustapha ◽  
Alla Kirshner ◽  
Danielle De Moissac ◽  
Lorrie A. Kirshenbaum
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Ventricular Myocytes ◽  
Vital Staining ◽  
Fold Increase ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Cytoplasmic Factor ◽  
Tnf Α ◽  
Factor Α

Nuclear factor-κB (NF-κB) is a ubiquitously expressed cellular factor regulated by the cytoplasmic factor inhibitor protein κBα (IκBα). Activation of NF-κB by cytokines, including tumor necrosis factor-α (TNF-α), requires the phosphorylation and degradation of IκBα. An anti-apoptotic role for NF-κB has recently been suggested. In the present study, we ascertained whether death-promoting signals and apoptosis mediated by TNF-α are suppressed by NF-κB in postnatal ventricular myocytes. Stimulation of myocytes with TNF-α resulted in a 12.1-fold increase ( P < 0.01) in NF-κB-dependent gene transcription and DNA binding compared with controls. This was accompanied by a corresponding increase in the NF-κB target protein A20 as determined by Western blot analysis. Vital staining revealed that TNF-α was not cytotoxic to myocytes and did not provoke apoptosis. Adenovirus-mediated delivery of a nonphosphorylatable form of IκBα to inactivate NF-κB prevented TNF-α-stimulated NF-κB-dependent gene transcription and nuclear NF-κB DNA binding. Importantly, myocytes stimulated with TNF-α and defective for NF-κB activation resulted in a 2.2-fold increase ( P < 0.001) in apoptosis. To our knowledge, the data provide the first indication that a functional NF-κB signaling pathway is crucial for suppressing death-promoting signals mediated by TNF-α in ventricular myocytes.

scholarly journals Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

2007 ◽  
Vol 408 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Ulrike Böer ◽  
Julia Eglins ◽  
Doris Krause ◽  
Susanne Schnell ◽  
Christof Schöfl ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Leucine Zipper ◽  
Luciferase Reporter ◽  
Lithium Salts ◽  
Transactivation Domain ◽  
Camp Response Element ◽  
Basic Leucine Zipper ◽  
Response Element ◽  
Leucine Zipper Domain

The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium.

scholarly journals Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding

2019 ◽  
Vol 116 (18) ◽  
pp. 8859-8868 ◽  
Author(s):  
Fan He ◽  
Wade Borcherds ◽  
Tanjing Song ◽  
Xi Wei ◽  
Mousumi Das ◽  
...  
Keyword(s):  
Dna Binding ◽  
Posttranslational Modifications ◽  
Dynamic Control ◽  
Transactivation Domains ◽  
Binding Surface ◽  
N Terminus ◽  
Response To Stress ◽  
Stress Signals

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT–DBD interaction.

scholarly journals The role of thyroid hormone receptor DNA binding in negative thyroid hormone-mediated gene transcription

2008 ◽  
Vol 41 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Anne Wulf ◽  
Marianne G Wetzel ◽  
Maxim Kebenko ◽  
Meike Kröger ◽  
Angelika Harneit ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Dna Binding ◽  
Gene Transcription ◽  
Hormone Receptor ◽  
Target Genes ◽  
Thyroid Hormone Receptor ◽  
Point Mutations ◽  
Chimeric Protein ◽  
Functional Consequences ◽  
Glycerol 3 Phosphate Dehydrogenase

Thyroid hormone 3,3′,5-tri-iodothyronine (T3) regulates gene expression in a positive and negative manner. Here, we analyzed the regulation of a positively (mitochondrial glycerol-3-phosphate dehydrogenase) and negatively T3-regulated target gene (TSHα). Thyroid hormone receptor (TR) activates mGPDH but not TSH promoter fragments in a mammalian one-hybrid assay. Furthermore, we investigated functional consequences of targeting TR to DNA independent of its own DNA-binding domain (DBD). Using a chimeric fusion protein of the DBD of yeast transcription factor Gal4 with TR, we demonstrated a positive regulation of gene transcription in response to T3. T3-mediated activation of this chimeric protein is further increased after an introduction of point mutations within the DBD of TR. Moreover, we investigated the capacity of TR to negatively regulate gene transcription on a DNA-tethered cofactor platform. A direct binding of TR to DNA via its own DBD is dispensable in this assay. We investigated functional consequences of point mutations affecting different domains of TR. Our data indicate that the DBD of TR plays a key role in direct DNA binding on positively but not on negatively T3-regulated target genes. Nevertheless, the DBD is involved in mediating negative gene regulation independent of its capacity to bind DNA.

Tissue factor gene transcription in serum-stimulated fibroblasts is mediated by recruitment of c-Fos into specific AP-1 DNA-binding complexes

Biochemistry ◽  
1995 ◽  
Vol 34 (38) ◽  
pp. 12355-12362 ◽  
Author(s):  
Sara J. Felts ◽  
Elizabeth S. Stoflet ◽  
Christopher T. Eggers ◽  
Michael J. Getz
Keyword(s):  
Dna Binding ◽  
Tissue Factor ◽  
Gene Transcription ◽  
Factor Gene ◽  
Dna Binding Complexes

Functional Differences in GATA-1 Affinity for Double Versus Single GATA-1 Binding Sites Dictate Synergistic Versus Antagonistic Interactions of PU.1 and GATA-1 in Myeloid Gene Transcription.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1608-1608
Author(s):  
Jian Du ◽  
Dharmesh Vyas ◽  
Qing Xi ◽  
Steven J. Ackerman
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Gene Transcription ◽  
Binding Sites ◽  
Zinc Fingers ◽  
Specific Expression ◽  
High Affinity ◽  
Dual Versus ◽  
P2 Promoter ◽  
Dual Site

Abstract Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.

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