scholarly journals The C-terminal domain of ParB is critical for dynamic DNA binding and bridging interactions which condense the bacterial centromere

2017 ◽  
Author(s):  
Gemma L. M. Fisher ◽  
César L. Pastrana ◽  
Victoria A. Higman ◽  
Alan Koh ◽  
James A. Taylor ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Dna Binding ◽  
Dna Condensation ◽  
Dna Binding Domain ◽  
Dynamic Nature ◽  
Terminal Domain ◽  
Binding Interface ◽  
Nucleoprotein Complexes

SUMMARYThe ParB protein forms DNA bridging interactions aroundparSto form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring atparS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensationin vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formationin vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

scholarly journals The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Gemma LM Fisher ◽  
César L Pastrana ◽  
Victoria A Higman ◽  
Alan Koh ◽  
James A Taylor ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Dna Condensation ◽  
Structural Basis ◽  
Dna Binding Domain ◽  
Dynamic Nature ◽  
Terminal Domain ◽  
Binding Interface

The ParB protein forms DNA bridging interactions around parS to condense DNA and earmark the bacterial chromosome for segregation. The molecular mechanism underlying the formation of these ParB networks is unclear. We show here that while the central DNA binding domain is essential for anchoring at parS, this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB-GFP foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks in Bacillus subtilis.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation

2003 ◽  
Vol 374 (2) ◽  
pp. 423-431 ◽  
Author(s):  
Christopher D. DEPPMANN ◽  
Tina M. THORNTON ◽  
Fransiscus E. UTAMA ◽  
Elizabeth J. TAPAROWSKY
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activator Protein 1 ◽  
Basic Leucine Zipper ◽  
Activator Protein

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1768-1776 ◽  
Author(s):  
Xiaojiao Fan ◽  
Xu Zhang ◽  
Yuwei Zhu ◽  
Liwen Niu ◽  
Maikun Teng ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Response Regulator ◽  
Regulatory System ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Promoter Regions ◽  
Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

scholarly journals Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

2006 ◽  
Vol 188 (14) ◽  
pp. 5167-5176 ◽  
Author(s):  
Deborah R. Yoder-Himes ◽  
Lee Kroos
Keyword(s):  
Dna Binding ◽  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Cell Movement ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Consensus Binding Site ◽  
Multicellular Development

ABSTRACT The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Ω4400. To determine if FruA is a direct regulator of Ω4400 transcription, a combination of in vivo and in vitro experiments was performed. Ω4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter −35 region in vitro. Mutations between bp −86 and −77 greatly reduced binding. One of these mutations had been shown previously to reduce Ω4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Ω4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Ω4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp −80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp −86 and −77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain.

scholarly journals Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo

2012 ◽  
Vol 8 (9) ◽  
pp. e1002906 ◽  
Author(s):  
Clinton R. Paden ◽  
J. Craig Forrest ◽  
Scott A. Tibbetts ◽  
Samuel H. Speck
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain

scholarly journals Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain.

1993 ◽  
Vol 13 (10) ◽  
pp. 5943-5956 ◽  
Author(s):  
S Hardy ◽  
Y Kong ◽  
S F Konieczny
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Fibroblast Growth Factor ◽  
Negative Regulation ◽  
Dna Binding Domain ◽  
Fibroblast Growth ◽  
Threonine Residue

MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic activity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.

scholarly journals Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

2007 ◽  
Vol 189 (14) ◽  
pp. 4984-4993 ◽  
Author(s):  
Jason R. Wickstrum ◽  
Jeff M. Skredenske ◽  
Ana Kolin ◽  
Ding J. Jin ◽  
Jianwen Fang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcription Activation ◽  
In Vitro Transcription ◽  
Binding Domain ◽  
Receptor Protein ◽  
Dna Binding Domain ◽  
Dimerization Domain ◽  
Half Site

ABSTRACT The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

scholarly journals Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction

2001 ◽  
Vol 82 (10) ◽  
pp. 2341-2351 ◽  
Author(s):  
Kelly J. Woytek ◽  
Dhandapani Rangasamy ◽  
Cynthia Bazaldua-Hernandez ◽  
Mike West ◽  
Van G. Wilson
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Genome Replication ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Recognition Element ◽  
C Terminus

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

scholarly journals Multiple Functions of the Nonconserved N-Terminal Domain of Yeast TATA-Binding Protein

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 87-93
Author(s):  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein S ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Core Domain ◽  
Terminal Domain ◽  
Two Factors

Abstract The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization. Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins. For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects. By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP. Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region. Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo. It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s).

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