scholarly journals Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

2019 ◽  
Vol 20 (15) ◽  
pp. 3692 ◽  
Author(s):  
Muthusamy Ramakrishnan ◽  
Mingbing Zhou ◽  
Chunfang Pan ◽  
Heikki Hänninen ◽  
Kim Yrjälä ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Inverted Repeats ◽  
Dna Binding Domain ◽  
Mobility Shift ◽  
Transposition Frequency ◽  
Terminal Inverted Repeats ◽  
Transposition Activity ◽  
Highly Active ◽  
Gel Mobility Shift

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

scholarly journals A single amino acid change in CUP2 alters its mode of DNA binding.

1990 ◽  
Vol 10 (9) ◽  
pp. 4778-4787 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
W Dixon ◽  
T D Tullius ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Dna Binding Domain ◽  
Sequence Motifs ◽  
Wild Type ◽  
Mobility Shift ◽  
Single Amino Acid Change

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

scholarly journals Role of the IE62 Consensus Binding Site in Transactivation by the Varicella-Zoster Virus IE62 Protein

2010 ◽  
Vol 84 (8) ◽  
pp. 3767-3779 ◽  
Author(s):  
Kris White ◽  
Hua Peng ◽  
John Hay ◽  
William T. Ruyechan
Keyword(s):  
Dna Binding ◽  
Varicella Zoster Virus ◽  
Binding Site ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Varicella Zoster ◽  
Mobility Shift Assay

ABSTRACT The varicella-zoster virus (VZV) IE62 protein is the major transcriptional activator. IE62 is capable of associating with DNA both nonspecifically and in a sequence-specific manner via a consensus binding site (5′-ATCGT-3′). However, the function of the consensus site is poorly understood, since IE62 efficiently transactivates promoter elements lacking this sequence. In the work presented here, sequence analysis of the VZV genome revealed the presence of 245 IE62 consensus sites throughout the genome. Some 54 sites were found to be present within putative VZV promoters. Electrophoretic mobility shift assay (EMSA) experiments using an IE62 fragment containing the IE62 DNA-binding domain and duplex oligonucleotides that did or did not contain the IE62 consensus binding sequence yielded KD (equilibrium dissociation constant) values in the nanomolar range. Further, the IE62 DNA binding domain was shown to have a 5-fold-increased affinity for its consensus site compared to nonconsensus sequences. The effect of consensus site presence and position on IE62-mediated activation of native VZV and model promoters was examined using site-specific mutagenesis and transfection and superinfection reporter assays. In all promoters examined, the consensus sequence functioned as a distance-dependent repressive element. Protein recruitment assays utilizing the VZV gI promoter indicated that the presence of the consensus site increased the recruitment of IE62 but not Sp1. These data suggest a model where the IE62 consensus site functions to down-modulate IE62 activation, and interaction of IE62 with this sequence may result in loss or decrease of the ability of IE62 to recruit cellular factors needed for full promoter activation.

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending

1992 ◽  
Vol 12 (5) ◽  
pp. 2037-2042
Author(s):  
A M Nardulli ◽  
D J Shapiro
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Dna Bending ◽  
Binding Domain ◽  
Estrogen Response Element ◽  
Dna Binding Domain ◽  
Mobility Shift ◽  
Response Element ◽  
Estrogen Response ◽  
Dna Fragment

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

scholarly journals Smooth Muscle-Specific Genes Are Differentially Sensitive to Inhibition by Elk-1

2005 ◽  
Vol 25 (22) ◽  
pp. 9874-9885 ◽  
Author(s):  
Jiliang Zhou ◽  
Guoqing Hu ◽  
B. Paul Herring
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Binding Site ◽  
Serum Response Factor ◽  
Specific Protein ◽  
Binding Domain ◽  
Dominant Negative ◽  
Differential Sensitivity ◽  
Dna Binding Domain ◽  
Mobility Shift

ABSTRACT Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including telokin, SM22α, and smooth muscle α-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only telokin and SM22α, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of telokin and SM22α without affecting expression of smooth muscle α-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.

UV photoaffinity labeling of Tn3 transposase–DNA complexes: identification of DNA binding domains

1996 ◽  
Vol 42 (1) ◽  
pp. 46-59
Author(s):  
Geoffrey S. Gottlieb ◽  
Michael A. Fennewald
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Target Site ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Dna Binding Domain ◽  
Transposition Frequency ◽  
Terminal Domain

The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition. The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion. The primary aim of this study is to define the relationship between the structure of Tru3 transposase and its DNA binding functions. We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain. The 70-kDa N-terminal domain encompasses the IR sequence-specific binding domain, as well as a nonspecific DNA binding domain that has been previously described. We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA. We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein. Mutations in this region reduce transposition frequency to 30–40% of the wild type. These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR. Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain. This suggests that this region may be involved in target site selection.Key words: transposon, Tn3, DNA–protein cross-linking, UV cross-linking, transposase.

scholarly journals Structural studies of AraR from B. thetaiotaomicron

2014 ◽  
Vol 70 (a1) ◽  
pp. C205-C205
Author(s):  
Changsoo Chang ◽  
Christine Tesar ◽  
Dmitry Rodionov ◽  
Xiaoqing Li ◽  
Robert Jedrzejczak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Sole Source ◽  
Binding Domain ◽  
Department Of Energy ◽  
Environmental Research ◽  
Dna Binding Domain ◽  
Nudix Hydrolase ◽  
Uncharacterized Protein ◽  
Mobility Shift ◽  
Space Group

The NrtR family of bacterial transcription factors is characterized by an N-terminal Nudix hydrolase-like effector binding domain and a C-terminal DNA binding domain. A bioinformatics analysis of the NrtR family represented by uncharacterized protein BT0354 in Bacteroides thetaiotaomicron suggests that these regulators control the catabolic pathways for L-arabinose. Many bacteria use L-arabinose as the sole source of carbon energy. The L-arabinose utilization pathway and its transcriptional regulation have been studied for a long time in several model microorganisms. Here we provide biochemical and structural characterization of the novel arabinose-responsive regulator of NrtR family protein BT0354, L-arabinose regulator from B. thetaiotaomicron (BtAraR). The BtAraR DNA binding and the role of effector molecule L-arabinose were confirmed using electrophoretic mobility shift assays. We have solved the crystal structures of BtAraR for two apo forms, and complexes with L-arabinose and double-stranded DNA target. The apo-1 form was solved as two dimers/AU in the R3 space group at 2.35 Å, while the apo-2 form was solved as one monomer/AU in the I213 space group at 2.56 Å resolution. The L-arabinose and DNA complex structures were solved as a dimer/AU in the P21 space group at 1.95 Å resolution and the P23 space group at 3.05 Å resolution, respectively. The biological unit of this protein is a dimer while the N-terminal ligand binding domain of the monomer adopts a Nudix hydrolase-like fold and the C-terminal DNA binding domain is a winged helix-turn-helix. The DNA binding-releasing mechanism can be rationalized through the comparison and analyses of these structures. The apo and DNA bound structures are more similar compared to the L-arabinose-bound structure. The r.m.s. deviation for the apo and DNA bound structures is 1.13 Å, while that for apo and the L-arabinose-bound structures is 4.54 Å. Details about the DNA binding mode, L-arabinose binding and L-arabinose induced structural change will be presented. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

scholarly journals Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from drosophila melanogaster.

1996 ◽  
Vol 43 (4) ◽  
pp. 611-621 ◽  
Author(s):  
A Rusin ◽  
A Niedziela-Majka ◽  
G Rymarczyk ◽  
A Ozyhar
Keyword(s):  
Dna Binding ◽  
Dna Interaction ◽  
Binding Domain ◽  
Structural Basis ◽  
Dna Binding Domain ◽  
Affinity Tag ◽  
Mobility Shift ◽  
Ecdysteroid Receptor ◽  
Dna Binding Domains ◽  
Binding Domains

Two members of the nuclear receptor superfamily, EcR and Ultraspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone-the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development. In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains. Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30. Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16%. Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose. The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene.

scholarly journals Presence and significance of a R110W mutation in the DNA-binding domain of GCM2 gene in patients with isolated hypoparathyroidism and their family members

2010 ◽  
Vol 162 (2) ◽  
pp. 407-421 ◽  
Author(s):  
Neeraj Tomar ◽  
Hema Bora ◽  
Ratnakar Singh ◽  
Nandita Gupta ◽  
Punit Kaur ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Family Members ◽  
Binding Domain ◽  
Compound Heterozygous ◽  
Dna Binding Domain ◽  
Nucleotide Polymorphisms ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Mobility Shift Assay

ObjectiveGlial cells missing 2 (GCM2) gene encodes a parathyroid-specific transcription factor. We assessed GCM2 gene sequence in patients with isolated hypoparathyroidism (IH).DesignCase–control study.MethodsComplete DNA sequencing of the GCM2 gene including its exons, promoter, and 5′ and 3′ UTRs was performed in 24/101 patients with IH. PCR–restriction fragment length polymorphism was used to detect a novel R110W mutation in all 101 IH patients and 655 healthy controls. Significance of the mutation was assessed by electrophoretic mobility shift assay (EMSA) and nuclear localization on transfection.ResultsA heterozygous R110W mutation was present in DNA-binding domain in 11/101 patients (10.9%) and absent in 655 controls (P<10−7). Four of 13 nonaffected first-degree relatives for five of these index cases had R110W mutation. Four heterozygous single nucleotide polymorphisms were found in the 5′ region. One of the 11 patients with R110W also had T370M change in compound heterozygous form. Mutant R110W and T370M GCM2 proteins showed decreased binding with GCM recognition elements on EMSA indicating loss of function. Both wild-type and R110W mutant GCM2 proteins showed nuclear localization.ConclusionsThe present study indicates a significant association of R110W variant with IH. Absence of effect of heterozygous R110W mutation on DNA binding and presence of the same mutation in asymptomatic family members indicate that additional genetic (akin to T370M change) or nongenetic factors might contribute to the expression of diseases in IH. Alternatively, it is possible that association of R110W with IH could be due to linkage disequilibrium with the unidentified relevant genes in IH.

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