scholarly journals Entropic pressure controls oligomerization of Vibrio cholerae ParD2 antitoxin

2021 ◽  
Author(s):  
Gabriela Garcia-Rodriguez ◽  
Yana Girardin ◽  
Alexander N. Volkov ◽  
Ranjan Kumar Singh ◽  
Gopinath Muruganandam ◽  
...  
Keyword(s):  
Dna Binding ◽  
Hydrogen Bonds ◽  
Vibrio Cholerae ◽  
Streptococcus Agalactiae ◽  
Full Length ◽  
Dna Binding Domain ◽  
Salt Bridges ◽  
Relative Positioning ◽  
Intrinsically Disordered ◽  
Extended Operator

AbstractParD2 is the antitoxin component of the parDE2 toxin-antitoxin module from Vibrio cholerae and consists of an ordered DNA binding domain followed by an intrinsically disordered ParE-neutralizing domain. In absence of the C-terminal IDP domain, VcParD2 crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than hydrophobic contacts. In solution, oligomerization of the full-length protein is restricted to a stable, open 10-mer or 12-mer, likely as a consequence of entropic pressure from the IDP tails. The relative positioning of successive VcParD2 dimers mimics the arrangement of Streptococcus agalactiae CopG dimers on their operator and allows for an extended operator to wrap around the VcParD2 oligomer.

scholarly journals Entropic pressure controls the oligomerization of the Vibrio cholerae ParD2 antitoxin

2021 ◽  
Vol 77 (7) ◽  
Author(s):  
Gabriela Garcia-Rodriguez ◽  
Yana Girardin ◽  
Alexander N. Volkov ◽  
Ranjan Kumar Singh ◽  
Gopinath Muruganandam ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Vibrio Cholerae ◽  
Streptococcus Agalactiae ◽  
Intrinsically Disordered Protein ◽  
Dna Binding Domain ◽  
Salt Bridges ◽  
Relative Positioning ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Extended Operator

ParD2 is the antitoxin component of the parDE2 toxin–antitoxin module from Vibrio cholerae and consists of an ordered DNA-binding domain followed by an intrinsically disordered ParE-neutralizing domain. In the absence of the C-terminal intrinsically disordered protein (IDP) domain, V. cholerae ParD2 (VcParD2) crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than by hydrophobic contacts. In solution, oligomerization of the full-length protein is restricted to a stable, open decamer or dodecamer, which is likely to be a consequence of entropic pressure from the IDP tails. The relative positioning of successive VcParD2 dimers mimics the arrangement of Streptococcus agalactiae CopG dimers on their operator and allows an extended operator to wrap around the VcParD2 oligomer.

scholarly journals Intrinsically Disordered Regions of the DNA-Binding Domain of Human FoxP1 Facilitate Domain Swapping

2020 ◽  
Vol 432 (19) ◽  
pp. 5411-5429 ◽  
Author(s):  
Exequiel Medina ◽  
Pablo Villalobos ◽  
George L. Hamilton ◽  
Elizabeth A. Komives ◽  
Hugo Sanabria ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Domain Swapping ◽  
Dna Binding Domain ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Disordered Regions

scholarly journals Mad proteins contain a dominant transcription repression domain.

1996 ◽  
Vol 16 (10) ◽  
pp. 5772-5781 ◽  
Author(s):  
D E Ayer ◽  
C D Laherty ◽  
Q A Lawrence ◽  
A P Armstrong ◽  
R N Eisenman
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Binding Domain ◽  
Full Length ◽  
Dna Binding Domain ◽  
Interaction Domain ◽  
Transcription Repression ◽  
Helix Loop Helix ◽  
Repression Domain

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

scholarly journals Regulation of Cell Cycle Transcription Factor Swi4 through Auto-Inhibition of DNA Binding

1999 ◽  
Vol 19 (10) ◽  
pp. 6729-6741 ◽  
Author(s):  
Kristin Baetz ◽  
Brenda Andrews
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Domain ◽  
Terminal Region ◽  
Full Length ◽  
Intramolecular Interactions ◽  
Dna Binding Domain ◽  
C Terminus ◽  
Binding Factor

ABSTRACTInSaccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G1/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively. The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role. SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains. Swi4 binding to SCBs is restricted to the late M and G1phases, when Swi6 is localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle. This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system. We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6. However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA intrans. Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain. Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs. The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA.

The TAL1 Short Isoform Generated By PRMT1-Mediated Alternative RNA Splicing Promotes Erythroid Differentiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1249-1249
Author(s):  
Shuiling Jin ◽  
Ngoc-Tung Tran ◽  
Hairui Su ◽  
Suming Huang ◽  
Xinyang Zhao ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Splicing ◽  
Short Form ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Binding Domain ◽  
Full Length ◽  
Dna Binding Domain ◽  
Transcriptional Regulatory ◽  
Megakaryocyte Differentiation

Abstract The isoforms of key transcription factors in hematopoiesis such as TAL1, GATA1 and RUNX1 are generated through alternative RNA splicing regulated by the PRMT1-RBM15 axis (Zhang et al. 2015). The functions of short isoforms of GATA1 (GATA1s) and RUNX1 (RUNX1a) are well characterized, yet it is unknown how the short isoform of TAL1 (TAL1s) regulates hematopoiesis. In this presentation, we report that the short isoform of TAL1, i.e. TAL1s, is generated via alternative RNA splicing as detected by isoform specific real-time PCR reactions using RNA isolated from leukemia cell lines and primary human cord blood cells. RBM15, an RNA binding protein, which is involved in chromosome translocation to produce RBM15-MKL1 fusion protein in acute megakaryocytic leukemia, regulates the alternative RNA splicing of TAL1. RBM15 promotes the production of full-length TAL1 mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway favors the production of TAL1s. RBM15 directly binds to intronic regions on TAL1 pre-mRNA. Binding of RBM15 is responsible for recruiting SF3B1-associated RNA splicing complex. Given that PRMT1 senses the hypoxia status of hematopoietic cells, the changing of TAL1s/TAL1fl ratio by PRMT1 activity may be an adaptive response of hematopoietic cells to hypoxia status. The short form TAL1s still contains the helix-loop-helix DNA binding domain but not the N terminal regions upstream of the DNA binding domain. Thus, the TAL1s may act as a dominant negative mutant of the full-length TAL1fl to block TAL1fl-regulated transcription. We demonstrated that overexpression of TAL1s not the full-length TAL1promotes the erythroid differentiation of K562 cells. Although TAL1 gene is required for both erythroid and megakaryocyte differentiation at early stage of hematopoiesis, TAL1s does not promote megakaryocyte differentiation. Therefore, fine-tuning the TAL1 isoforms by the PRMT1-RBM15 axis determine the cell fate of a MEP progenitor cell. Using immunoprecipitation assays and mass spectrometry analysis, we identified proteins specifically associated with the N terminal region of TAL1. How unique TAL1s-associated transcriptional regulatory complex plays in erythroid differentiation will be discussed in the presentation in comparison with the Tal1fl-asociated protein complex. In summary, our findings stratify another new layer of regulation by PRMT1, which relays extracellular signals (such as hypoxia signal) to transcriptional regulatory program. Given that PRMT1 is often constitutively highly expressed in leukemia cells, how overproduction of short form TAL1 interferes with normal hematopoiesis may help to explain the molecular mechanisms of many hematological malignancies associated with dysregulation of TAL1 expression. Disclosures No relevant conflicts of interest to declare.

DNA-binding regulates site-specific ubiquitination of IRF-1

2013 ◽  
Vol 449 (3) ◽  
pp. 707-717 ◽  
Author(s):  
Vivien Landré ◽  
Emmanuelle Pion ◽  
Vikram Narayan ◽  
Dimitris P. Xirodimas ◽  
Kathryn L. Ball
Keyword(s):  
Dna Binding ◽  
E3 Ligase ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Docking Site ◽  
Interacting Protein ◽  
Site Specific ◽  
E3 Ligases ◽  
Intrinsically Disordered ◽  
C Terminus

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

scholarly journals Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

2021 ◽  
Author(s):  
Bonnie L. Bassler ◽  
Olivia Duddy ◽  
Xiuliang Huang ◽  
Justin Silpe
Keyword(s):  
Dna Binding ◽  
Quorum Sensing ◽  
Vibrio Cholerae ◽  
Dna Sequences ◽  
Genetic Screen ◽  
Binding Domain ◽  
Communication Process ◽  
Dna Binding Domain ◽  
Phage Gene ◽  
Small Regulatory Rna

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven exclusively by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain as necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA-binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter.

scholarly journals Evolution of human, chicken, alligator, frog and zebrafish mineralocorticoid receptors: Allosteric influence on steroid specificity

2017 ◽  
Author(s):  
Yoshinao Katsu ◽  
Kaori Oka ◽  
Michael E. Baker
Keyword(s):  
Dna Binding ◽  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Full Length ◽  
Dna Binding Domain ◽  
Mineralocorticoid Receptors ◽  
Terminal Domain ◽  
Terrestrial Vertebrate ◽  
Hinge Domain

AbstractWe studied the response to aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, corticosterone, progesterone, 19-norprogesterone and spironolactone of human, chicken, alligator, frog and zebrafish full-length mineralocorticoid receptors (MRs) and truncated MRs, lacking the N-terminal domain (NTD) and DNA-binding domain (DBD), in which the hinge domain and ligand binding domain (LBD) were fused to a GAL4-DBD. Compared to full-length MRs, some vertebrate MRs required higher steroid concentrations to activate GAL4-DBD-MR-hinge/LBD constructs. For example, 11-deoxycortisol activated all full-length vertebrate MRs, but did not activate truncated terrestrial vertebrate MRs and was an agonist for truncated zebrafish MR. Progesterone, 19-norProgesterone and spironolactone did not activate full-length and truncated human, alligator and frog MRs. However, at 10 nM, these steroids activated full-length chicken and zebrafish MRs; at 100 nM, these steroids had little activity for truncated chicken MRs, while retaining activity for truncated zebrafish MRs, evidence that regulation of progestin activation of chicken MR resides in NTD/DBD and of zebrafish MR in hinge-LBD. Zebrafish and chicken MRs contain a serine corresponding to Ser810 in human MR, required for its antagonism by progesterone, suggesting novel regulation of progestin activation of chicken and zebrafish MRs. Progesterone may be a physiological activator of chicken and zebrafish MRs.

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