SEARCH · PubMed Health

Results for “nucleoprotein complex”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources

PubMed1991

Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation.

The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.

Adenosine Triphosphate↗

PubMed1986

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction.

DNA Repair↗

PubMed1983

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes.

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r less than 0.1-0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.

Animals↗

PubMed1991

Role of lipopolysaccharide and IL-4 in control of transcription of the class II A alpha gene.

The class II (Ia) MHC molecules are cell surface proteins that regulate the activation of T cells. B lymphocyte expression of class II molecules has been shown to be influenced by a number of external stimuli. It has been previously demonstrated that treatment of these cells with IL-4 leads to an increase in class II gene transcription at 18 h as well as to an increase in steady state class II mRNA. It has also been previously demonstrated that LPS treatment of splenic B cells from athymic mice results in a decrease in steady state mRNA encoding the A alpha class II protein. This decrease persists for at least 18 h. Nuclear run-on transcription assays now demonstrate that although steady state mRNA levels for A alpha are decreased by LPS treatment of athymic mouse lymphocytes, LPS does not decrease A alpha gene transcription, but rather modestly activates transcription of this class II gene. LPS and IL-4 have been demonstrated to be synergistic stimuli for a number of genes. Costimulation of splenic lymphocytes from athymic mice with IL-4 plus LPS leads to activation of transcription, but the increase in transcription is no more than that seen with IL-4 stimulation alone. However, in costimulated lymphocytes, steady state A alpha-encoding mRNA levels are intermediate between the increased levels seen with IL-4 stimulation and the decreased levels seen with LPS stimulation. Therefore, LPS and IL-4 act nonsynergistically in class II gene transcription and the effects of LPS in decreasing steady state mRNA are most likely posttranscriptional. An IL-4-inducible and an LPS-inducible DNA-binding protein have been previously identified in splenic lymphocytes from athymic mice. Both nuclear binding proteins form complexes with the same DNA fragments from a control region of the A alpha gene. These nucleoprotein complexes comigrate under nondenaturing conditions and display identical patterns of binding with a panel of oligonucleotide competitors. Oligonucleotides representing protein binding sites of the IL-4 and LPS-induced DNA-binding proteins cross-compete for protein binding. Therefore, the binding proteins induced by LPS and IL-4 are likely related, and may function at different efficiencies as activators of A alpha gene transcription.

Animals↗

PubMed1984

Assignment of resonances of exchangeable protons in the NMR spectrum of the complex formed by Escherichia coli ribosomal protein L25 and uniformly nitrogen-15 enriched 5 S RNA fragment.

The downfield proton NMR spectrum of the aqueous nucleoprotein complex formed by Escherichia coli ribosomal protein L25 and uniformly nitrogen-15 enriched 5 S RNA fragment is presented. Many proton resonances show the effects of scalar coupling to nitrogen-15 and these resonances are assigned to nucleic acid imino protons. Selective nitrogen-15 decoupling difference proton spectroscopy revealed nitrogen-15 and proton chemical shift correlations from which the base types of nucleic acid imino proton resonances could be assigned because the nitrogen-15 chemical shifts of nucleic acid guanine and uracil imino nitrogens have separate small ranges for both nucleoproteins and isolated nucleic acids.

Escherichia coli↗

PubMed1989

Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure.

Seizure causes a rapid and protracted increase in transcription factor AP-1 levels in the brain. The composition of AP-1 nucleoprotein complexes changes with time after seizure as a result of the sequential appearance and disappearance of Fos and several Fos-related proteins. Although these changes occur over an 8 hr time period, they are triggered by 15 min of seizure. Alterations in the levels and composition of transcription factors may represent one of the molecular mechanisms underlying neuronal adaptation.

Animals↗

PubMed1994

The use of monoclonal antibodies for studying intermediates in DNA repair by the Escherichia coli Uvr(A)BC endonuclease.

The Escherichia coli Uvr(A)BC endonuclease acts in a progression of several distinct steps accompanied by changes in the conformation of macromolecular constituents, the overall architecture of the complex, and its stoichiometry. In order to probe these structural changes, we generated monoclonal antibodies (mAbs) to Uvr proteins. The anti-UvrA mAb, A2D1, recognizing the N-terminal zinc-finger region of UvrA, and the anti-UvrB mAb, B2E2, having an epitope within the 43 C-terminal amino acids of UvrB, were purified and further characterized. It was found that A2D1 mAb interacts in solution both with UvrA-UvrB and UvrA-DNA complexes in the presence of the requisite ATP. This implies that the N-terminal zinc-finger of UvrA doesn't play a direct role in its interactions with UvrB and DNA. On the other hand, A2D1 does inhibit formation of the UvrB-damaged DNA preincision complex, apparently by preventing UvrB delivery by UvrA. The interaction of B2E2 with UvrA-UvrB and nucleoprotein complexes, including UvrB, suggests that the highly hydrophobic C-terminal domain of UvrB (i) doesn't participate in its interaction with UvrA, (ii) is accessible to this mAb in an intermediate UvrA-UvrB DNA helix-tracking complex, and (iii) seems to be directly involved in the formation of the preincision complex. These conclusions are supported by the finding that the neutralizing effect of A2D1 and B2E2 on the Uvr(A)BC endonuclease is significantly decreased if the preincision complex is preformed prior to mAbs addition.

Antibodies, Monoclonal↗

PubMed1998

Differential silver-staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nonisotopic method for characterizing gel-separated histone-DNA complexes.

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

Animals↗

PubMed1993

Protein-protein interactions in gene regulation: the cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor.

Maximal repression by the CytR protein depends on the formation of nucleoprotein complexes in which CytR interacts with DNA and with cAMP-cAMP receptor protein (CRP). Here we demonstrate that CytR regulates transcription from deoP2 promoters in which the entire CytR recognition sequence has been eliminated. Furthermore, CytR proteins deleted for the DNA-binding domain repress deoP2 in vivo and interact with deoP2 in vitro in a strictly cAMP-CRP-dependent fashion. These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CytR-DNA interactions may primarily serve to stabilize the nucleo-protein complex. This type of specificity mechanism may represent a general concept in the recruitment of DNA-binding proteins in combinatorial regulatory systems.

Amino Acid Sequence↗

PubMed2006

Structural aspects of interactions within the Myc/Max/Mad network.

Recently determined structures of a number of Myc family proteins have provided significant insights into the molecular nature of complex assembly and DNA binding. These structures illuminate the details of specific interactions that govern the assembly of nucleoprotein complexes and, in doing so, raise more questions regarding Myc biology. In this review, we focus on the lessons provided by these structures toward understanding (1) interactions that govern transcriptional repression by Mad via the Sin3 pathway, (2) homodimerization of Max, (3) heterodimerization of Myc-Max and Mad-Max, and (4) DNA recognition by each of the Max-Max, Myc-Max, and Mad-Max dimers.

Animals↗

PubMed1993

Differential nuclear expression of enhancer A DNA-binding proteins in human first trimester trophoblast cells.

In order to investigate the possible molecular regulatory mechanisms that repress classical HLA class I and stimulate nonclassical HLA-G and E-class I gene transcription in human trophoblast cells, we searched for the nuclear expression of the enhancer A DNA-binding proteins of the KBF1/NF-KB/rel family. Using both purified extravillous cytotrophoblast and villous syncytiotrophoblast from first trimester human placenta, it appeared that members of this family were present in the cytotrophoblast and absent in the syncytiotrophoblast. First, using the double stranded enhancer A DNA nucleotidic sequence that contains the palindromic KB site, known to be the binding site of the p50 subunits of KBF1-NF-KB and c-rel factors, we demonstrated, by band-shift assay, that binding activity, inhibited by addition of anti-p50 polyclonal serum, was present in cytotrophoblast as well as control maternal decidual cells, embryonic fibroblasts, and the trophoblast-derived JAR cell line. In contrast, this DNA-protein complex was undetectable in syncytiotrophoblast nuclear extracts. The specificity of this protein-DNA complex was further demonstrated by its disappearance upon competition with an excess of cold homologous nucleotidic competitor. Other nucleoprotein complexes were also detected in all nuclear extracts, including syncytiotrophoblast, that were competed out by an excess of cold enhancer A competitor DNA but were not affected by the addition of anti-p50 or anti-NF-KB sera, suggesting the presence of additional enhancer A-binding factors different from the KBF1/NF-KB/rel family. Second, using a Western immunoblot analysis, a doublet around 85 kDa was specifically stained by the same anti-p50 serum in cytotrophoblast, maternal decidual cells, embryonic fibroblasts, and the JAR nuclear extracts whereas no signal was obtained in syncytiotrophoblast. Finally, immunofluorescence cell staining using the same anti-p50 serum showed a positive staining in both cytoplasm and nucleus of cytotrophoblast and its absence in syncytiotrophoblast. We hypothesize that this enhancer A DNA-binding factor might represent the c-rel trans-acting factor, related to p50/KBF1/NF-KB proteins, and we discuss its possible relevance to the HLA class I transcription in human tissues.

Base Sequence↗

PubMed2006

Vif-deficient HIV reverse transcription complexes (RTCs) are subject to structural changes and mutation of RTC-associated reverse transcription products.

Reverse transcription (RTn) in HIV-infected cells occurs in a nucleoprotein complex termed the reverse transcription complex (RTC). RTCs containing RT activity and integrase (IN) were shown to be heterogeneous in size and density on sucrose velocity and equilibrium gradients. WT and Vif-deficient (Deltavif) RTCs produced by infection with virus from permissive cells displayed similar sedimentation characteristics, while RTCs from Deltavif virus produced in non-permissive cells demonstrated a reduction in the major RTC form and more of the RTn products in rapidly sedimenting structures. APOBEC3G derived from virions did not co-sediment with RTCs, but RTCs from Deltavif infections showed elevated levels of mutations in RTn products, consistent with APOBEC3G and other mutational mechanisms. The most mutated transcripts were present within rapidly sedimenting RTCs. Thus, virus without functional vif, produced from non-permissive cells, forms abnormal RTCs that contain increased mutation of RTC-associated RTn products in newly infected target cells.

APOBEC-3G Deaminase↗

PubMed1987

Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.

Bacterial Proteins↗

PubMed1991

Supercoiling is essential for the formation and stability of the initiation complex at the divergent malEp and malKp promoters.

malEp and malKp are divergent and partially overlapping promoters of the Escherichia coli maltose regulon, whose activity depends on the presence of two transcriptional activators. MalT and CRP (cAMP receptor protein). Their activation involves a common 210 base-pair regulatory region encompassing multiple binding sites for both activators. Using a supercoiled plasmid containing malEp and malKp as template, purified proteins and a single-round transcription assay, we developed an in vitro system in which both promoters behave as in vivo. In this system, malEp and malKp are active only in the presence of both MalT and CRP, and various mutations in the MalT or CRP binding sites affect the promoters in the same way as they do in vivo. We showed that supercoiling plays a crucial role not only for the formation of the initiation complex at malEp and malKp but also for its stability. In addition, dimethylsulphate protection experiments provide evidence that the nucleoprotein complexes formed by CRP and MalT bound to malEp and malKp on supercoiled and relaxed DNA are different. We speculate that one of the roles of supercoiling might be to assist the assembly of a preinitiation complex involving the regulatory region DNA and several molecules of MalT and CRP.

Bacterial Proteins↗

PubMed1979

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.

Animals↗

PubMed1994

Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli.

The Escherichia coli hns gene encodes the abundant nucleoid-associated DNA-binding protein H-NS. Mutations in hns alter the expression of many genes with unrelated functions and result in a derepression of the proU operon (proVWX) without abolishing the osmotic control of its transcription. We have investigated the interactions of H-NS with the proU regulatory region by deletion analysis of cis-acting sequences, competitive gel retardation assays, and DNase I footprinting. The negative effect of H-NS on proU transcription was mediated by cis-acting sequences within proV but did not depend on the presence of a curved DNA segment upstream of the proU-35 region previously characterized as a target for H-NS binding in vitro. We detected a 46-base pair high affinity H-NS binding region downstream of the proU promoter at the 5' end of the proV gene and a complex array of additional H-NS binding sites which suggest the presence of an extended H-NS nucleoprotein complex. Most of the H-NS binding sites were highly A+T-rich and carried stretches of 5 or more consecutive A-T base pairs. The implications of our results for the osmotic regulation of proU transcription are discussed.

Amino Acid Transport Systems↗

PubMed2000

Relationship of oligomerization to DNA binding of Wheat dwarf virus RepA and Rep proteins.

Members of the genus Mastrevirus (family Geminiviridae) produce a complementary-sense (c-sense) transcription unit with the potential to encode two proteins, RepA and Rep. In the present work, we have studied the DNA-protein complexes formed by the Wheat dwarf virus (WDV) RepA protein within the WDV large intergenic region. WDV RepA forms large nucleoprotein complexes near the TATA boxes of the viral complementary-sense and virion-sense (v-sense) promoters (the RepA C- and V-complexes, respectively), a location similar to those of WDV Rep-DNA complexes but with distinct DNase I footprints. We have also studied the relationship of oligomerization of WDV RepA and Rep proteins to DNA-protein complex formation. Using chemical cross-linking, we have determined that both WDV proteins can form oligomers in solution. Interestingly, the pH is critical for the monomer-oligomer equilibrium and small changes produce a displacement in such a way that at pH </= 7.0, the predominant species is an octamer while at pH >/= 7.4 it is a monomer. Complex formation is also strongly affected by pH and occurs more efficiently at pH 7.0-7.4. We found that preformed oligomers interact very poorly with DNA. Thus, our data are consistent with a stepwise model for protein-DNA complex assembly in which monomers interact with DNA and then with other monomers to assemble an oligomeric structure on the DNA. These results may be relevant for studies on the DNA binding, replication, and transcription properties of geminivirus proteins.

Base Sequence↗

PubMed1986

Preferential nucleosome placement on pBR322 restriction fragments.

Two restriction fragments of DNA containing the regulatory feature GTG/CAC were experimentally associated with core histones. The reconstituted DNA-histone complexes consisted of different forms of mononucleosomes. Lambda exonuclease and Fnu4HI were used to probe the structure of each distinct nucleoprotein complex. For each of the DNA fragments, one form of particle was produced that showed preferred placement of the core octamer on the DNA. The GTG/CAC base triplets may play some role in determining the final histone core positions in these reconstitutes.

Base Sequence↗