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At least 613 records · Page 34Linked to original sources

PubMed1994

Identification of cyclic AMP response element in the human renin gene.

In order to identify the mechanism by which cyclic AMP stimulates expression of the human renin gene (REN), the effect of forskolin was tested in transient expression analyses of REN 5'-flanking DNA-chloramphenicol acetyltransferase (CAT) reporter gene constructs in secondary cultures of human chorio-decidual cells, a major site of renin synthesis. Forskolin induced a mean 5-fold stimulation which was localized to DNA in the region -249 to -162 with respect to the transcription start site (+1). Such DNA also mediated a response to forskolin in heterologous (HSV thymidine kinase) promoter constructs. Strong cAMP-response element (CRE) homology at -222 to -218 resembled the target for members of the CRE binding protein (CREB) family. Gel shift assays demonstrated similarly migrating nucleoprotein complexes for oligonucleotides containing the putative REN CRE as for a canonical CRE, in chorio-decidual, JEG-3 and HeLa nuclear extracts. Mutation of residues critical for CREB attachment reduced binding. In conclusion, a CRE was identified at -222 to -218 that appears critical for cAMP-induced human renin gene transcription.

Animals↗

PubMed1994

Tumour necrosis factor alpha and interleukin-1 beta induce specific subunits of NFKB to bind the HIV-1 enhancer: characterisation of transcription factors controlling human immunodeficiency virus type 1 gene expression in neural cells.

In human astrocytoma and neuroblastoma cell lines tumour necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta) induced NFKB and an additional KB-specific protein of approximately 80 K to bind the HIV-1 enhancer. One nucleoprotein complex contained prototypical NFKB comprising of p50 and p65 subunits and a second contained the p65 subunit and an 80 K factor, p80. Over a 24 hr period of cytokine stimulation the p50/p65 complex of NFKB was present in the nucleus whilst the second complex declined in abundance after two hours due to the decline of p80. In unstimulated cells, DNAse I footprinting revealed a previously unidentified octamer-like binding site in the negative regulatory element (NRE) of the HIV-1 long terminal repeat (LTR) which specifically bound protein factors present in human astrocytoma, neuroblastoma and murine oligodendroglioma cell lines. A second unique motif, also in the NRE, was observed with extracts from one human neuroblastoma cell line and a murine oligodendroglioma. Footprinting analysis also confirmed that Sp1, TATA, Site A and Site B motifs of the LTR were occupied by nuclear factors present in neural cells.

Astrocytoma↗

PubMed1996

Transcriptional repression activity of N-MYC protein requires phosphorylation by MAP kinase.

The transrepressing function of the N-myc protein is due to the distinct domains located at the N-terminus. In this report we introduced various point mutations around the myc boxes of the N-myc protein to examine whether the phosphorylation of the protein affected its transrepressing function. Serine (Ser) residues located at amino acid numbers 12, 31, 51, and 65 were changed to leucine or arginine, and the expression vectors of the mutant proteins were transfected to HeLa cells together with the luciferase gene linked to the MHC class I gene. Among the mutants, only the N(51)-myc carrying mutation at Ser(51), a target for mitogen-activated protein kinase (MAP kinase), lost the repression activity, while the other mutant proteins preserved it. Formation in vitro of the specific nucleoprotein complexes on the H2TF1/NFkappaB element, a major target for transrepression by N-myc protein, was interfered by the wild-type N-myc protein, but not by the Ser(51)-mutated protein. The results suggest that the phosphorylation of the N-myc protein at Ser(51) by MAP kinase is required for the transcriptional repression activity of the protein.

Amino Acid Sequence↗

PubMed1997

Telomere-telomere interactions and candidate telomere binding protein(s) in mammalian sperm cells.

We have used fluorescent in situ hybridization to localize telomeres within the nuclei of sperm from six mammals (human, rat, mouse, stallion, boar, and bull). In minimally swollen sperm of mouse and rat, most of the telomeres are clustered within a limited area in the posterior part of nuclei. In sperm of other species, telomeres associate into tetrameres and dimers. On swelling of sperm cells with heparin/dithiotriethol, telomere associations disperse, and hybridization signals become smaller in size and their numbers approach or correspond to the number of chromosome ends in a haploid genome. Quantitation of telomere loci indicates that dimeric associations are prominent features of mammalian sperm nuclear architecture. Higher order telomere-telomere interactions and organization develop during meiotic stages of human spermatogenesis. At this stage, telomeres also become associated with the nuclear membrane. In an attempt to elucidate the molecular mechanisms underlying telomere interactions in sperm, we have identified a novel protein activity that binds to the double-stranded telomeric repeat (TTAGGG)n. Sperm telomere binding protein(s) (STBP) was extracted from human and bull sperm by 0.5 M NaCl. STBP does not bind single-stranded telomeric DNA and is highly specific for single base substitutions in a duplex DNA sequence. Depending on the conditions of binding, we observed the formation of several nucleoprotein complexes. We have shown that there is a transition between complexes, which indicates that the slower migrating complex is a multimer of the higher mobility one. We propose that STBP participates in association between the telomere domains which were microscopically observed in mammalian spermatozoa.

Animals↗

PubMed1998

Hmg4, a new member of the Hmg1/2 gene family.

High mobility group (HMG) proteins are abundant components of mammalian nuclei and fall into three families. The members of one such family, HMG1 and HMG2, are ubiquitously expressed and facilitate the formation of nucleoprotein complexes where the DNA is sharply bent. We have identified a mouse cDNA that codes for a novel 200-amino-acid protein of the HMG1/2 family, which we called HMG4. The mouse Hmg4 gene is highly expressed in the embryo; Hmg4 transcripts are barely detectable in adult tissues. The human HMG4 gene, which is extremely similar to its mouse homolog, has been sequenced as part of chromosome X, band q28. HMG4, HMG1, and HMG2 proteins have been highly conserved during vertebrate evolution, suggesting that each has at least some unique property. It is possible that HMG4 is required during development.

Amino Acid Sequence↗

PubMed1993

Lrp, a global regulatory protein of Escherichia coli, binds co-operatively to multiple sites and activates transcription of ilvIH.

Lrp (Leucine-responsive regulatory protein) has recently been recognized as a major regulatory protein that controls the expression of many operons in Escherichia coli. Footprinting and gel retardation experiments with DNA from ilvIH, one of the operons controlled positively by Lrp, indicate that Lrp binds to six sites over a 200 base-pair region upstream from the promoter. Binding of Lrp to some of these sites is highly co-operative. We suggest a consensus sequence for Lrp binding based upon a comparison of six binding sites. An analysis of mutants indicates that five out of six binding sites are important for transcription activation and that two or three adjacent Lrp binding sites act synergistically in vivo. The observed synergistic effects in vivo may result from co-operative binding of Lrp to adjacent sites. We propose a model in which multiple binding sites contribute to the formation of a nucleoprotein complex, but only a particular proximal site positions Lrp properly so that it interacts with RNA polymerase.

Acetolactate Synthase↗

PubMed1995

Poly(dA).poly(dT) forms very stable nucleosomes at higher temperatures.

The synthetic polymer poly(dA).poly(dT) was long thought to be refractory to nucleosome formation. Several years ago our laboratory demonstrated that the polymer could be mixed with authentic nucleosomes in a low-salt exchange procedure to form a nucleoprotein complex that behaved in a manner identical with that of nucleosomes. Competitive exchange assays at 37 degrees C showed that the homopolymer reconstituted about as well as heterogenous-sequence DNA. However, studies by other laboratories have shown that the conformation of poly(dA).poly(dT) depends on temperature; the polymer converts from its well-known, atypical structure, found at ambient temperature, to a conformation more closely resembling a canonical B form as temperature is increased. We have measured the ability of the homopurine.homopyrimidine to form nucleosomes as a function of temperature. It is seen that poly(dA).poly(dT) forms nucleosomes more strongly as the temperature of the exchange mixture is increased, so that poly(dA).(dT) outcompetes heterogeneous-sequence DNA for histones at elevated temperatures.

Animals↗

PubMed1997

The Mu transposase tetramer is inactive in unassisted strand transfer: an auto-allosteric effect of Mu A promotes the reaction in the absence of Mu B.

A tetramer of the Mu transposase is the structural and functional core in all three stable higher-order nucleoprotein complexes (Type 0, Type 1 and Type 2 transpososomes) generated in a defined in vitro strand transfer reaction. Although functional in donor cleavage, we report here that contrary to previous belief, the Mu A tetramer is incapable of unassisted strand transfer. The Mu B protein is required to stimulate the tetramer for intermolecular strand transfer. In the absence of Mu B protein we show that additional Mu A molecules must be added to the core tetramer to stimulate intramolecular strand transfer. Mapping experiments indicate that domain II of the assisting Mu A mediates functional interactions with the core tetramer. The recipient site for Mu A stimulated strand transfer on the A tetramer is likely in domain II and is clearly different from the domain IIIb site used by the Mu B protein. The Mu accessory end binding sites and the Mu enhancer are not required in the Mu A assisted strand transfer, suggesting that helper A molecules in solution can interact with the core tetramer to stimulate the reaction. Finally, we argue that the strand transfer activity and protein sites for target interaction reside within the core tetramer; hence the role of the stimulatory A molecules appears to be limited to that of an auto-allosteric effector.

Allosteric Regulation↗

PubMed1998

Possible roles of nucleocapsid protein of MoMuLV in the specificity of proviral DNA synthesis and in the genetic variability of the virus.

Retroviral nucleocapsid (NC) protein, in addition to its structural roles in the virion core, is involved in the early and late phases of the viral replication cycle. To further characterise the role of NC protein of MoMuLV (NCp10) in the replication of the viral genome, the influence of NCp10 on self-primed versus primer-specific reverse transcription has been analysed in vitro. The results show that NCp10 can enhance the specificity of proviral DNA synthesis by inhibiting self-primed cDNA synthesis while promoting primer-specific DNA synthesis within active NCp10-RNA nucleoprotein complexes. Retroviruses are known to show a high degree of variability and this prompted us to examine the possible implication of NCp10 in the genetic variability of MoMuLV. The ability of reverse transcriptase (RT) to extend different mutated primers using an RNA or a DNA template has been investigated in the presence or in the absence of NCp10. NCp10 was found to have different effects on RT depending on the nature of the template: an enhancement at the elongation level of mutated primers using RNA as template versus a slight inhibition using DNA as template. These observations suggest that NCp10 could be implicated in the genetic variability of MoMuLV by allowing nucleotide misincorporation principally during minus strand DNA synthesis.

3T3 Cells↗

PubMed1998

pyrH-encoded UMP-kinase directly participates in pyrimidine-specific modulation of promoter activity in Escherichia coli.

The carAB operon of the enterics Escherichia coli K-12 and Salmonella typhimurium LT2, encoding the sole carbamoylphosphate synthetase (CPSase) of these organisms, is transcribed from two promoters in tandem, carP1 upstream and carP2 downstream, repressed respectively by pyrimidines and arginine. We present evidence that the pyrH gene product (the hexameric UMP-kinase) directly participates in the pyrimidine-specific control of carP1 activity. Indeed, we have isolated in E. coli a particular type of pyrH mutation (pyrH41) that retains a quasi-normal UMP-kinase activity, but yet is impaired in the pyrimidine-specific repression of the P1 promoter of the carAB operon of E. coli and of S. typhimurium. Moreover, the pyrimidine-dependent inhibition of in vivo Dam methylase modification of adenine -106 upstream of the carP1 promoter is altered in this pyrH mutant. The recessive pyrH41 allele bears a single C-G to A-T transversion that converts alanine 94 into glutamic acid (A94E). Although overexpression of pyrH41 results in UMP-kinase levels far above that of a wild-type strain, pyrimidine-specific repression of the carAB operon is not restored under these conditions. Similarly, overexpression of the UMP-CMP-kinase gene of Dictyostelium discoideum in the pyrH41 mutant does not restore pyrimidine-mediated control of carP1 promoter activity, in spite of the elevated UMP-kinase activity measured in such transformants. These results indicate that besides its catalytic function in the de novo pyrimidine biosynthesis, E. coli UMP-kinase fulfils an additional, but previously unrecognized role in the regulation of the carAB operon. UMP-kinase might function as the real sensor of the internal pyrimidine nucleotide pool and act in concert with the integration host factor (IHF) and aminopeptidase A (PepA alias CarP and XerB) in the elaboration of the complex nucleoprotein structure required for pyrimidine-specific repression of carP1 promoter activity.

Adenine↗

PubMed1998

Molecular interactions between the coactivator CBP and the human T-cell leukemia virus Tax protein.

The oncoprotein Tax, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high-level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Recent evidence suggests that Tax stimulates HTLV-I transcription through recruitment of the cellular coactivator protein CBP to the HTLV-I promoter, promoting high-level viral replication via the transcriptional activation properties associated with CBP. Tax directly contacts the KIX domain of CBP to stably anchor the coactivator to nucleoprotein complexes at the promoter. Here, we identify KIX amino acid residues 588 to 683 as the minimal region sufficient for interaction with Tax. This region is similar to the minimal KIX amino acid residues necessary for strong interaction with phosphorylated CREB, and is composed of a structural domain that forms an extensive hydrophobic core. We further show that a double point mutation in KIX differentially affects the binding of Tax and phosphorylated CREB, suggesting that these transcription factors may recognize unique amino acid residues within the KIX domain. These observations suggest that Tax directly contacts the hydrophobic core of KIX, and provides a structural framework to further define the molecular interactions between Tax and CBP.

CREB-Binding Protein↗

PubMed1998

Binding of phage phi29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage Phi29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase alpha subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low affinity to a site centered at position -39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position -71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase alpha-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high affinity. A mutational analysis revealed that the site at -71 is critical for a stable interaction between protein p4 and RNA polymerase, and for efficient repression, whereas mutation of the site at -39 had only a small effect on repression efficiency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.

Bacillus subtilis↗

PubMed1998

Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces.

The pi35.0 protein of plasmid R6K regulates transcription and replication by binding a DNA sequence motif (TGAGR) arranged either asymmetrically into 22 bp direct repeats (DRs) in the gamma origin, or symmetrically into inverted half-repeats (IRs) in the operator of its own gene, pir. The binding patterns of the two natural forms of the pi protein and their heterodimers revealed that the predominant species, pi35.0 (35.0 kDa), can bind to a single copy of the DR as either a monomer or a dimer while pi30.5 (30.5 kDa) binds only as a dimer. We demonstrate that only one subunit of a pi35.0 dimer makes specific contact with DNA. Electron microscopic (EM) analysis of the nucleoprotein complexes formed by pi35.0 and DNA fragments containing all seven DRs revealed coupled ("hand-cuffed") DNA molecules that are aligned in a parallel orientation. Antiparallel orientations of the DNA were not observed. Thus, hand-cuffing depends on a highly ordered oligomerization of pi35.0 in such structures. The pi protein (pi35.0, pi30.5) binds to an IR as a dimer or heterodimer but not as a monomer. Moreover, a single amino acid residue substitution, F200S (pir200), introduced into pi30.5 severely destabilizes dimers of this protein in solution and concomitantly prevents binding of this protein to the IR. This mutation also changes the stability of pi35.0 dimers but it does not change the ability of pi35.0 to bind IRs. To explain these observations we propose that the diverse interactions of pi variants with DNA are controlled by multiple surfaces for protein oligomerization.

Amino Acid Sequence↗

PubMed1999

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

Binding Sites↗

PubMed2000

Coordinated control of XerC and XerD catalytic activities during Holliday junction resolution.

Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons. In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time. Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner. This is associated with a reciprocal impairment of the recombinase's own ability to initiate catalysis. The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect. We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase. The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome.

Amino Acid Substitution↗

PubMed2000

Selection of homeotic proteins for binding to a human DNA replication origin.

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.

Amino Acid Sequence↗

PubMed2000

The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal.

The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous "HMG-box" DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.

Amino Acid Sequence↗

PubMed2001

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo.

Capsid↗