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.

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.

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 Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

2015 ◽  
Vol 197 (16) ◽  
pp. 2675-2684 ◽  
Author(s):  
Seram Nganbiton Devi ◽  
Brittany Kiehler ◽  
Lindsey Haggett ◽  
Masaya Fujita
Keyword(s):  
Bacillus Subtilis ◽  
Mutant Protein ◽  
Phosphoryl Group ◽  
Atp Binding ◽  
Histidine Kinases ◽  
Content Type ◽  
Point Mutant ◽  
Terminal Domain

ABSTRACTEntry into sporulation inBacillus subtilisis governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur intrans. To test this hypothesis, we generated a series ofB. subtilisstrains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in atransfashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressedin vivo. Taken together, thesein vitroandin vivoresults reinforce the evidence that KinA autophosphorylation is able to occur in atransfashion.IMPORTANCEAutophosphorylation of histidine kinases is known to occur by either thecis(one subunit of kinase phosphorylating itself within the multimer) or thetrans(one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided directin vivoandin vitroevidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur intranswithin the homotetramer complex. While the physiological and mechanistic significance of thetransautophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.

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.

scholarly journals A Region of Bacillus subtilis CodY Protein Required for Interaction with DNA

2005 ◽  
Vol 187 (12) ◽  
pp. 4127-4139 ◽  
Author(s):  
Pascale Joseph ◽  
Manoja Ratnayake-Lecamwasam ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Dna Recognition ◽  
Transcriptional Regulators ◽  
Site Directed Mutagenesis ◽  
Regulation Of Transcription ◽  
Gram Positive Bacteria ◽  
Target Promoters

ABSTRACT Bacillus subtilis CodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G+C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.

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 Two Regions of GerE Required for Promoter Activation in Bacillus subtilis

2002 ◽  
Vol 184 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Dinene L. Crater ◽  
Charles P. Moran
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
In Vitro Transcription ◽  
Dna Binding Domain ◽  
Promoter Activation ◽  
Transcription Activators

ABSTRACT GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

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