scholarly journals PRDM9 forms a trimer by interactions within the zinc finger array

2019 ◽  
Vol 2 (4) ◽  
pp. e201800291
Author(s):  
Theresa Schwarz ◽  
Yasmin Striedner ◽  
Andreas Horner ◽  
Karin Haase ◽  
Jasmin Kemptner ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Correlation Spectroscopy ◽  
Binding Studies ◽  
Mobility Shift ◽  
Fluorescence Correlation ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays ◽  
Dna Complex ◽  
Zinc Finger Array

PRDM9 is a trans-acting factor directing meiotic recombination to specific DNA-binding sites by its zinc finger (ZnF) array. It was suggested that PRDM9 is a multimer; however, we do not know the stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and characterized with electrophoretic mobility shift assays, mass spectrometry, and fluorescence correlation spectroscopy the stoichiometry of the PRDM9 multimer of two different murine PRDM9 alleles carrying different tags and domains produced with different expression systems. Based on the migration distance of the PRDM9–DNA complex, we show that PRDM9 forms a trimer. Moreover, this stoichiometry is adapted already by the free, soluble protein with little exchange between protein monomers. The variable ZnF array of PRDM9 is sufficient for multimerization, and at least five ZnFs form already a functional trimer. Finally, we also show that only one ZnF array within the PRDM9 oligomer binds to the DNA, whereas the remaining two ZnF arrays likely maintain the trimer by ZnF–ZnF interactions.

scholarly journals TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution

2005 ◽  
Vol 387 (2) ◽  
pp. 401-409 ◽  
Author(s):  
Jolanta KOPEC ◽  
Alexander BERGMANN ◽  
Gerhard FRITZ ◽  
Elisabeth GROHMANN ◽  
Walter KELLER
Keyword(s):  
Amino Acids ◽  
Broad Host Range ◽  
Mobility Shift ◽  
Gram Positive ◽  
Nick Site ◽  
Α Helix ◽  
Electrophoretic Mobility Shift Assays ◽  
Dna Complex ◽  
Β Sheet

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase–oriT complex.

scholarly journals Control of Directionality in Bacteriophage mv4 Site-Specific Recombination: Functional Analysis of the Xis Factor

2009 ◽  
Vol 192 (3) ◽  
pp. 624-635 ◽  
Author(s):  
Michèle Coddeville ◽  
Paul Ritzenthaler
Keyword(s):  
Binding Sites ◽  
Basic Protein ◽  
Lactobacillus Delbrueckii ◽  
Mobility Shift ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the host attB site during Lactobacillus delbrueckii lysogenization. The mv4 prophage is excised during the induction of lytic growth. Excisive site-specific recombination between the attR and attL sites is also catalyzed by the phage-encoded recombinase, but the directionality of the recombination is determined by a second phage-encoded protein, the recombination directionality factor (RDF). We have identified and functionally characterized the RDF involved in site-specific excision of the prophage genome. The mv4 RDF, mv4Xis, is encoded by the second gene of the early lytic operon. It is a basic protein of 56 amino acids. Electrophoretic mobility shift assays demonstrated that mv4Xis binds specifically to the attP and attR sites via two DNA-binding sites, introducing a bend into the DNA. In vitro experiments and in vivo recombination assays with plasmids in Escherichia coli and Lactobacillus plantarum demonstrated that mv4Xis is absolutely required for inter- or intramolecular recombination between the attR and attL sites. In contrast to the well-known phage site-specific recombination systems, the integrative recombination between the attP and attB sites seems not to be inhibited by the presence of mv4Xis.

scholarly journals The PAX6 gene is activated by the basic helix–loop–helix transcription factor NeuroD/BETA2

2003 ◽  
Vol 376 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Eleonora MARSICH ◽  
Amedeo VETERE ◽  
Matteo DI PIAZZA ◽  
Gianluca TELL ◽  
Sergio PAOLETTI
Keyword(s):  
Transcription Factor ◽  
Pax6 Gene ◽  
Mobility Shift ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Dna Binding Sites ◽  
E Boxes ◽  
Nih 3T3 ◽  
Electrophoretic Mobility Shift Assays

PAX6 is a transcription factor that plays an important role during pancreatic morphogenesis. The aim of the present study is to identify the upstream activator(s) of the PAX6 gene possibly involved in the early stages of pancreatic differentiation. Recently, individual elements regulating PAX6 gene activity in the pancreas have been identified in a 1100 bp Spe/HincII fragment 4.6 kb upstream of exon 0. Preliminary sequence analysis of this region revealed some potential DNA-binding sites (E boxes) specific for the binding of basic helix–loop–helix transcription factors. By using electrophoretic mobility shift assays, we demonstrated that both nuclear protein extracts from insulin-secreting RINm5F cells and in vitro-translated NeuroD/BETA2 can bind specifically to these E boxes. Furthermore, by transient transfection experiments we demonstrated that the expression of basic helix–loop–helix transcription factor NeuroD/BETA2 can induce activation of the PAX6 promoter in the NIH-3T3 cell line. Thus we show that NeuroD/BETA2 is involved in the activation of the expression of PAX6 through E boxes in the PAX6 promoter localized in a 1.1 kb sequence within the 4.6 kb untranslated region upstream of exon 0.

scholarly journals Variably Sized and Multi-Colored Silica-Nanoparticles Characterized by Fluorescence Correlation Methods for Cellular Dynamics

Materials ◽  
2020 ◽  
Vol 14 (1) ◽  
pp. 19
Author(s):  
Chan-Gi Pack ◽  
Bjorn Paulson ◽  
Yeonhee Shin ◽  
Min Kyo Jung ◽  
Jun Sung Kim ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Correlation Spectroscopy ◽  
Cell Uptake ◽  
Core Shell ◽  
Therapeutic Effects ◽  
Ferrite Core ◽  
Shell Nanoparticles ◽  
Fluorescence Correlation ◽  
Correlation Methods

Controlling the uptake of nanoparticles into cells so as to balance therapeutic effects with toxicity is an essential unsolved problem in the development of nanomedicine technologies. From this point of view, it is useful to use standard nanoparticles to quantitatively evaluate the physical properties of the nanoparticles in solution and in cells, and to analyze the intracellular dynamic motion and distribution of these nanoparticles at a single-particle level. In this study, standard nanoparticles are developed based on a variant silica-based nanoparticle incorporating fluorescein isothiocyanate (FITC) or/and rhodamine B isothiocyanate (RITC) with a variety of accessible diameters and a matching fluorescent cobalt ferrite core-shell structure (Fe2O4/SiO2). The physical and optical properties of the nanoparticles in vitro are fully evaluated with the complementary methods of dynamic light scattering, electron microscopy, and two fluorescence correlation methods. In addition, cell uptake of dual-colored and core/shell nanoparticles via endocytosis in live HeLa cells is detected by fluorescence correlation spectroscopy and electron microscopy, indicating the suitability of the nanoparticles as standards for further studies of intracellular dynamics with multi-modal methods.

scholarly journals The Regulator of Nitrate Assimilation in Ascomycetes Is a Dimer Which Binds a Nonrepeated, Asymmetrical Sequence

1998 ◽  
Vol 18 (3) ◽  
pp. 1339-1348 ◽  
Author(s):  
Joseph Strauss ◽  
M. Isabel Muro-Pastor ◽  
Claudio Scazzocchio
Keyword(s):  
Nitrate Assimilation ◽  
Binding Studies ◽  
Binding Ability ◽  
Zinc Cluster ◽  
First Case ◽  
Vitro Binding ◽  
Nitrate Induction ◽  
Dna Complex

ABSTRACT The regulation of nitrate assimilation seems to follow the same pattern in all ascomycetes where this process has been studied. We show here by in vitro binding studies and a number of protection and interference techniques that the transcription factor mediating nitrate induction in Aspergillus nidulans, a protein containing a binuclear zinc cluster DNA binding domain, recognizes an asymmetrical sequence of the form CTCCGHGG. We further show that the protein binds to its consensus site as a dimer. We establish the role of the putative dimerization element by its ability to replace the analogous element of the cI protein of phage λ. Mutagenesis of crucial leucines of the dimerization element affect both the binding ability of the dimer and the conformation of the resulting protein-DNA complex. This is the first case to be described where a dimer recognizes such an asymmetrical nonrepeated sequence, presumably by each monomeric subunit making different contacts with different DNA half-sites.

scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

Interaction of GAL4 and GAL80 gene regulatory proteins in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3446-3451
Author(s):  
N F Lue ◽  
D I Chasman ◽  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Single Step ◽  
Regulatory Proteins ◽  
Mobility Shift ◽  
Ura3 Gene ◽  
Upstream Activation Sequence ◽  
Chromatographic Procedure ◽  
Gene Regulatory ◽  
Dna Complex ◽  
Activation Sequence

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

scholarly journals Transcriptional regulation of Mycobacterium tuberculosis hupB gene expression

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1637-1647 ◽  
Author(s):  
Satya Deo Pandey ◽  
Mitali Choudhury ◽  
Manjula Sritharan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Iron Homeostasis ◽  
Iron Concentration ◽  
Dna Interaction ◽  
Intracellular Iron ◽  
Mobility Shift ◽  
Dna Footprinting ◽  
Sequence Of Events ◽  
Electrophoretic Mobility Shift Assays

The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR–Fe2+ complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml−1; 0.36 µM Fe) was still detectable at 8 µg Fe ml−1 (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml−1 (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein–DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

scholarly journals The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner

2008 ◽  
Vol 190 (12) ◽  
pp. 4351-4359 ◽  
Author(s):  
Thomas J. Goss
Keyword(s):  
Klebsiella Pneumoniae ◽  
Upstream Region ◽  
Mobility Shift ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Sensitive Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Knockout Mutation

ABSTRACT The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

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