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Results for “nucleoprotein complex”

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PubMed1977

Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.

Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5')-Gm, was oxidized with sodium periodate to convert the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5'termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end of mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes.

Animals↗

PubMed1993

Identification of a compact DNA-binding domain in the gene 5 protein of Pf1 bacteriophage.

The structure of the gene 5 protein of filamentous bacteriophage Pf1 and its interaction with viral DNA have been investigated by a series of limited proteolysis experiments. The ability of purified proteolytic fragments of the Pf1 gene 5 protein to bind oligonucleotides and polynucleotides was monitored by gel retardation and fluorescence. The results show the presence of a compact DNA-binding "core" domain consisting of residues 1-112 of the protein, which is protected from proteolysis in the nucleoprotein complex. Digestion of the free gene 5 protein with subtilisin produces a smaller fragment (residues 7-102) which can no longer bind DNA. Although the N-terminal "core" domain shows full DNA binding activity by fluorescence, the gel retardation experiments suggest reduced kinetic stability of this domain in complexes with oligonucleotides, resulting from the removal of residues 113-144 from the C-terminus of the protein. The sequence of the C-terminal 32 amino acid residues is unusual, with a high proportion of alanine, glutamine, and proline residues which may be related to the role of this sequence in stabilizing the complex.

Amino Acid Sequence↗

PubMed1984

DNA replication of simian virus 40 chromatin in digitonin-treated and saponin-treated permeable cells.

Permeable cell systems have been developed by treatment of simian virus 40 (SV40)-infected cells with digitonin or saponin for studying in vitro DNA replication of chromatin. SV40 DNA replicated efficiently in these systems in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. Analysis of the time course of the replicated SV40 DNA I, II and replicating intermediates by gel electrophoresis and autoradiography indicated that SV40 DNA replicated completely in the form of nucleoprotein complexes (SV40 chromatin).

Animals↗

PubMed2006

Retroviral DNA integration: reaction pathway and critical intermediates.

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.

Blotting, Western↗

PubMed1993

Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.

The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.

Bacteriophage M13↗

PubMed1999

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein.

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Adenosine Triphosphate↗

PubMed2002

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent.

We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDSPAGE separation of p66 and p51 polypeptides, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.

Amino Acid Sequence↗

PubMed1980

Saccharomyces cerevisiae plasmid, Scp or 2 mum: intracellular distribution, stability and nucleosomal-like packaging.

Cell fractions from yeast strains known to harbor the plasmic 2 mum or Scp were treated with nucleases used to probe eukaryotic chromosome structure. Scp and subfragments were identified by hybridization to natural or cloned Scp probes according to Southern (34). Specificity was confirmed with non-Scp probes. Copy/haploid nuclear genome(n) was estimated from reconstructions at a resolution of 0.5/n. About 43-67% of the total cellular copy exists as nucleoprotein complexes which separate from other debris on isokinetic sucrose gradients with s-values of 67-110. These complexes are totally degraded by DNAase I. Digestion with micrococcal nuclease produced integral-sized fragments; they are not generated by direct mixing of pure Scp with nuclear chromatin from a[cir] strain. Initial digests gave a repeat of 168 +/- 3 base pairs (bp) for both Scp and nuclear nucleoprotein; advanced digests reduced the nuclear repeat relative to Scp by 8 bp. Of a potential 37 repeat units/plasmid, 31-32 were directly measured. A strain difference in Scp autodegradation was found. A partial nuclease resistant form was also demonstrated whose abundance was cell strain and growth stage dependent. Both Scp isomers exist in these complexes which are structurally similar to simian viral 40 minichromosomes.

Deoxyribonuclease I↗

PubMed1994

Protection of mice against an influenza virus infection by oral vaccination with viral nucleoprotein incorporated into immunostimulating complexes.

Influenza A virus nucleoprotein (NP) was integrated into immunostimulating complexes (ISCOMs) after attachment of bacterial lipopolysaccharide to the antigen. Oral immunization with these NP-ISCOMs protected mice fully against an otherwise lethal challenge infection with an unrelated influenza virus subtype without the appearance of severe clinical signs or extensive pathological lesions in the lungs. Mice immunized with analogous bovine serum albumine-incorporated ISCOMs all died. After oral immunization, high titers of NP-specific antibodies, particularly IgA, could be detected in the bronchoalveolar fluid and in the blood serum. No cytotoxic lymphocytes could be demonstrated in the spleens or the lungs of vaccinated mice, and no anti-NP antibody-dependent cytolysis of infected host cells was mediated by complement or in the form of an antibody-dependent cell cytotoxicity. However, a vigorous delayed-type hypersensitivity reaction was produced after probing vaccinated animals with purified NP. No comparable protective immunity or antibody response was induced by a strictly intragastric administration of NP-ISCOMs. It appears, therefore, that the general and local immune response in the lungs was primarily stimulated through contact of NP-ISCOMs with the mucous membrane of the oro-pharyngeal cavity and that cytotoxic effects did not play a major role for the establishment of the protective immunity. Partial protection against a lethal challenge was observed in chickens immunized with NP-ISCOMs in the drinking water.

Administration, Oral↗

PubMed1994

Use of an inducible site-specific recombinase to probe the structure of protein-DNA complexes involved in F plasmid partition in Escherichia coli.

The induced expression of a tightly regulated site-specific recombinase is shown to efficiently form intracellular DNA rings of well-defined nucleotide sequences in Escherichia coli. To provide information on the organization of an intracellular protein-DNA complex, the linking number distributions of excised DNA rings containing cognate binding sites for the protein can be measured after their isolation. Application of this approach to the partition system of the E. coli F plasmid suggests that the SopB protein and the sopC locus, the latter being composed of 12 tandemly joined imperfect repeats of a 43 base-pair motif, form a complex in which the DNA is wrapped right-handedly around a multimeric protein core; the presence of a single copy of a 43 base-pair motif on a DNA appears to be sufficient to nucleate the formation of this nucleoprotein complex.

Bacterial Proteins↗

PubMed1992

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.

Base Sequence↗

PubMed1994

HMG domain proteins: architectural elements in the assembly of nucleoprotein structures.

The high-mobility group (HMG) domain is a DNA-binding motif that is shared abundant non-histone components of chromatin and by specific regulators of transcription and cell differentiation. The HMG family of proteins comprises members with multiple HMG domains that bind DNA with low sequence specificity, and members with single HMG domains that recognize specific nucleotide sequences. Common properties of HMG domain proteins include interaction with the minor groove of the DNA helix, binding to irregular DNA structures, and the capacity to modulate DNA structure by bending. DNA bending induced by the HMG domain can facilitate the formation of higher-order nucleoprotein complexes, suggesting that HMG domain proteins may have an architectural role in assembling such complexes.

Amino Acid Sequence↗

PubMed1998

Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor.

The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26 degrees C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23 degrees C. The histone-like nucleoid structuring protein H-NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H-NS or a factor regulated by H-NS bound within the pap regulatory region at 23 degrees C but not at 37 degrees C, as evidenced by H-NS-dependent inhibition of methylation of the pap GATC sites designated GATC-I and GATC-II. These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H-NS bound specifically to a pap DNA fragment containing the GATC-I and GATC-II sites. Moreover, H-NS blocked methylation of these pap GATC sites in vitro: H-NS blocked pap GATC methylation at 1.4 microM but was unable to do so at higher concentrations at which non-specific binding occurred. Thus, non-specific binding of H-NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H-NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H-NS with pap regulatory DNA. We hypothesize that a function of H-NS such as oligomerization was altered at 23 degrees C, which enabled H-NS to repress pap gene expression through the formation of a specific nucleoprotein complex.

Artificial Gene Fusion↗

PubMed2001

DNA binding by the ETS-domain transcription factor PEA3 is regulated by intramolecular and intermolecular protein.protein interactions.

The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly.

Amino Acid Sequence↗

PubMed1976

Release of RNA--DNA-protein complex during differentiation of the water mould Allomyces arbuscula.

A correlation between release of a nucleic acid protein complex into the external medium and sporangial differentiation was found. Conditions preventing differentiation also stopped the release. Lethal lysis is not involved in this release process. Extracellular nucleic acids are very heterogenous, consisting of nucleotides as well as acid-precipitable nucleic acids. RNA was found to be associated with proteins and a hetero-duplex RNA--DNA associated with this nucleoprotein. Some speculations are presented about possible correlation between the release of nucleoprotein complexes and the intracellular events of differentiation.

Cell Differentiation↗

PubMed2001

Functional organization of single and paired V(D)J cleavage complexes.

RAG-1 and RAG-2 initiate V(D)J recombination by binding to specific recognition sequences (RSS) and then cleave the DNA in two steps: nicking and hairpin formation. Recent work has established that a dimer of RAG-1 and either one or two monomers of RAG-2 bind to a single RSS, but the enzymatic contributions of the RAG molecules within this nucleoprotein complex and its functional organization have not been elucidated. Using heterodimeric protein preparations containing both wild-type and catalytically deficient RAG-1 molecules, we found that one active monomer is sufficient for both nicking and hairpin formation at a single RSS, demonstrating that a single active site can carry out both cleavage steps. Furthermore, the mutant heterodimers efficiently cleaved both RSS in a synaptic complex. These results strongly suggest that two RAG-1 dimers are responsible for RSS cleavage in a synaptic complex, with one monomer of each dimer catalyzing both nicking and hairpin formation at each RSS.

Binding Sites↗

PubMed1995

Interaction of normal and mutant SRY proteins with DNA.

In mammals, sex determination is caused by the Y-chromosome gene SRY. The DNA-binding domain of human SRY protein is similar to those of the chromatin protein HMG1. Like HMG1, SRY binds to kinked DNA structures, and bends linear DNA sharply upon binding. We analysed the biochemical properties of mutant SRY proteins from five patients with complete gonadal dysgenesis: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally, and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence, thus discriminating different target sites. We suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY in chromatin is essential for the expression of genes involved in testis differentiation.

Base Sequence↗

PubMed1994

Dynamics of the nucleoprotein structure of simian virus 40 regulatory region during viral development.

The regulatory region of SV40 is composed of multiple elements, including the origin of replication (ori), the encapsidation signal (ses) and the enhancer. Here, the structure of the chromatin and nucleoprotein complexes in a region encompassing ses and part of the enhancer was investigated in detail by in situ probing with DNase I. We have used a model experimental system based on plasmids which carry parts of the SV40 regulatory region. The results demonstrate that a specific nucleoprotein structure at the region is formed early after transfection. The overall structure is maintained throughout the viral life cycle. The observed DNase digestion pattern is consistent with the presence of a mixed population of viral minichromosomes with various, but not random, nucleosomal arrangements in that region. Specific modulations, which are associated with the various stages of the viral life cycle, are superimposed on the general structure. The most dramatic changes occur at nucleotides 34 and 113, located at both ends of ses and flanking the GC-box region. Some of the changes depend on the presence of viral gene product(s), probably a late (capsid) protein. The results further suggest that the condensed minichromosome within the viral particle assumes a highly specific configuration in this region. The nucleoprotein structure is sensitive to modifications of the primary nucleotide sequence and to flanking DNA elements. There is good correlation between distortions in the nucleoprotein structure and the inability of mutant plasmids to be packaged, substantiating the requirement for proper chromatin condensation in viral packaging.

Animals↗