scholarly journals Sequential XylS-CTD Binding to the Pm Promoter Induces DNA Bending Prior to Activation

2010 ◽  
Vol 192 (11) ◽  
pp. 2682-2690 ◽  
Author(s):  
Patricia Domínguez-Cuevas ◽  
Juan-Luís Ramos ◽  
Silvia Marqués
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Independent Manner ◽  
Form Complex ◽  
Terminal Domain ◽  
Proximal Site ◽  
Footprint Analysis ◽  
Global Curvature ◽  
Polymerase Binding

ABSTRACT XylS protein, a member of the AraC family of transcriptional regulators, comprises a C-terminal domain (CTD) involved in DNA binding and an N-terminal domain required for effector binding and protein dimerization. In the absence of benzoate effectors, the N-terminal domain behaves as an intramolecular repressor of the DNA binding domain. To date, the poor solubility properties of the full-length protein have restricted XylS analysis to genetic approaches in vivo. To characterize the molecular consequences of XylS binding to its operator, we used a recombinant XylS-CTD variant devoid of the N-terminal domain. The resulting protein was soluble and monomeric in solution and activated transcription from its cognate promoter in an effector-independent manner. XylS binding sites in the Pm promoter present an intrinsic curvature of 35° centered at position −42 within the proximal site. Gel retardation and DNase footprint analysis showed XylS-CTD binding to Pm occurred sequentially: first a XylS-CTD monomer binds to the proximal site overlapping the RNA polymerase binding sequence to form complex I. This first event increased Pm bending to 50° and was followed by the binding of the second monomer, which further increased the observed global curvature to 98°. This generated a concomitant shift in the bending center to a region centered at position −51 when the two sites were occupied (complex II). We propose a model in which DNA structure and binding sequences strongly influence XylS binding events previous to transcription activation.

scholarly journals NAD-Dependent DNA-Binding Activity of the Bifunctional NadR Regulator of Salmonella typhimurium

1999 ◽  
Vol 181 (2) ◽  
pp. 648-655 ◽  
Author(s):  
Thomas Penfound ◽  
John W. Foster
Keyword(s):  
Dna Binding ◽  
Integral Membrane Protein ◽  
Binding Activity ◽  
Consensus Sequences ◽  
Dna Binding Activity ◽  
Nicotinamide Mononucleotide ◽  
Regulator Protein ◽  
Footprint Analysis ◽  
Polymerase Binding

ABSTRACT NadR is a 45-kDa bifunctional regulator protein. In vivo genetic studies indicate that NadR represses three genes involved in the biosynthesis of NAD. It also participates with an integral membrane protein (PnuC) in the import of nicotinamide mononucleotide, an NAD precursor. NadR was overexpressed and purified as a His-tagged fusion in order to study its DNA-binding properties. The protein bound to DNA fragments containing NAD box consensus sequences. NAD proved to be the relevant in vivo corepressor, but full NAD dependence of repressor activity required nucleotide triphosphates. DNA footprint analysis and gel shift assays suggest that NadR binds as a multimer to adjacent NAD boxes. The DNA-repressor complex would sequester a potential RNA polymerase binding site and thereby decrease expression of thenad regulon.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

scholarly journals Reduced sulphydryl groups are required for DNA binding of Ku protein

1993 ◽  
Vol 293 (3) ◽  
pp. 769-774 ◽  
Author(s):  
W W Zhang ◽  
M Yaneva
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Binding Activity ◽  
Heat Inactivation ◽  
Cysteine Residues ◽  
Dna Binding Activity ◽  
Sulphydryl Groups ◽  
Ku Protein

The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.

scholarly journals The nucleoid as a scaffold for the assembly of bacterial signaling complexes

2017 ◽  
Author(s):  
Audrey Moine ◽  
Leon Espinosa ◽  
Eugenie Martineau ◽  
Mutum Yaikhomba ◽  
P J Jazleena ◽  
...  
Keyword(s):  
Dna Binding ◽  
Independent Manner ◽  
Signaling Systems ◽  
Bacterial Signaling ◽  
Aminoacid Sequence ◽  
Membrane Bound ◽  
Gliding Bacterium ◽  
Positively Charged

ABSTRACTThe FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.AUTHOR SUMMARYIn this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the Frz kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

scholarly journals Physical interactions between Gsx2 and Ascl1 regulate the balance between progenitor expansion and neurogenesis in the mouse lateral ganglionic eminence

2019 ◽  
Author(s):  
Kaushik Roychoudhury ◽  
Joseph Salomone ◽  
Shenyue Qin ◽  
Masato Nakafuku ◽  
Brian Gebelein ◽  
...  
Keyword(s):  
Dna Binding ◽  
Ventricular Zone ◽  
Luciferase Assay ◽  
Proximity Ligation Assay ◽  
Ganglionic Eminence ◽  
Independent Manner ◽  
Helix Loop Helix ◽  
Lateral Ganglionic Eminence ◽  
Physical Interactions

AbstractThe Gsx2 homeodomain transcription factor is required to maintain neural progenitor identity in the lateral ganglionic eminence (LGE) within the developing ventral telencephalon, despite its role in upregulating the neurogenic factor Ascl1. How Gsx2 maintains cells as progenitors in the presence of a pro-differentiation factor is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in dividing subapical progenitors within the LGE ventricular zone (VZ). Moreover, we show that while Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors that do not express Gsx2, co-expression of Gsx2 with Ascl1 inhibits neurogenesis in these cells. To investigate the mechanisms underlying this inhibition, we used a cell-based luciferase assay to show that Gsx2 reduced the ability of Ascl1 to activate target gene expression in a dose-dependent and DNA binding-independent manner. Yeast 2-hybrid and co-immunoprecipitation assays revealed that Gsx2 physically interacts with the basic-Helix-Loop-Helix (bHLH) domain of Ascl1, and DNA binding assays demonstrated that this interaction interferes with the ability of Ascl1 to form homo- or heterodimers with E-proteins such as Tcf3 on DNA. To further assess for in vivo molecular interactions between these transcription factors within the telencephalon, we modified a proximity ligation assay for embryonic tissue sections and found that Ascl1:Gsx2 interactions are enriched within VZ progenitors, whereas Ascl1:Tcf3 interactions predominate in basal progenitors. Altogether, these findings suggest that physical interactions between Gsx2 and Ascl1 limit Ascl1:Ascl1 and Ascl1:Tcf3 interactions, and thereby inhibit Ascl1-dependennt neurogenesis and allow for progenitor expansion within the LGE.

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).

scholarly journals Single stranded DNA annealing is a conserved activity of telomere resolvases

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246212
Author(s):  
Siobhan L. McGrath ◽  
Shu Hui Huang ◽  
Kerri Kobryn
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Bacterial Species ◽  
Dna Binding Protein ◽  
Tyrosine Recombinase ◽  
Single Stranded Dna ◽  
Terminal Domain ◽  
Topoisomerase Ib ◽  
Replication Intermediates

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA’s annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

scholarly journals Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus

2020 ◽  
Author(s):  
Sruti DebRoy ◽  
Victor Aliaga Tobar ◽  
Gabriel Galvez ◽  
Srishtee Arora ◽  
Xiaowen Liang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Pathogenic Bacteria ◽  
Group A Streptococcus ◽  
Protein A ◽  
Data Availability ◽  
Human Pathogen ◽  
Transcript Levels ◽  
Catabolite Control Protein A ◽  
Group A

SummaryCatabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in non-pathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIPseq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIPseq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.Data sharing and data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

scholarly journals Pf1, a Novel PHD Zinc Finger Protein That Links the TLE Corepressor to the mSin3A-Histone Deacetylase Complex

2001 ◽  
Vol 21 (13) ◽  
pp. 4110-4118 ◽  
Author(s):  
Gregory S. Yochum ◽  
Donald E. Ayer
Keyword(s):  
Dna Binding ◽  
Histone Deacetylase ◽  
Zinc Finger Protein ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
Binding Domain ◽  
Independent Manner ◽  
Alpha Helices ◽  
Interacting Protein

ABSTRACT The mSin3A-histone deacetylase corepressor is a multiprotein complex that is recruited by DNA binding transcriptional repressors. Sin3 has four paired amphipathic alpha helices (PAH1 to -4) that are protein-protein interaction motifs and is the scaffold upon which the complex assembles. We identified a novel mSin3A-interacting protein that has two plant homeodomain (PHD) zinc fingers we term Pf1, for PHD factor one. Pf1 associates with mSin3A in vivo and recruits the mSin3A complex to repress transcription when fused to the DNA binding domain of Gal4. Pf1 interacts with Sin3 through two independent Sin3 interaction domains (SIDs), Pf1SID1 and Pf1SID2. Pf1SID1 binds PAH2, while Pf1SID2 binds PAH1. Pf1SID1 has sequence and structural similarity to the well-characterized 13-amino-acid SID of the Mad bHLHZip repressor. Pf1SID2 does not have sequence similarity with either Mad SID or Pf1SID1 and therefore represents a novel Sin3 binding domain. Mutations in a minimal fragment of Pf1 that encompasses Pf1SID1 inhibited mSin3A binding yet only slightly impaired repression when targeted to DNA, implying that Pf1 might interact with other corepressors. We show that Pf1 interacts with a mammalian homolog of the Drosophila Groucho corepressor, transducin-like enhancer (TLE). Pf1 binds TLE in an mSin3A-independent manner and recruits functional TLE complexes to repress transcription. These findings suggest that Pf1 may serve to bridge two global transcription networks, mSin3A and TLE.

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