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PubMed1980

Transcription of reconstituted simian virus 40 nucleoprotein complexes.

The regulatory effects of host cellular histones on the transcription of simian virus 40 (SV40) DNA were investigated by using reconstituted and native SV40 nucleoprotein complexes (NPCs). Reconstituted NPCs were prepared from SV40 DNA and the combination fraction of five histones, H1, H2A, H2B, H3, and H4, isolated from the nuclei of permissive (CV-1) or nonpermissive (BALB/c 3T3, rat liver, and calf thymus) cells. Native NPCs were prepared by alkali disruption of purified SV40 virions. Nuclease digestion of these NPCs gave regular patterns of bands similar to those of SV40 NPCs from SV40-infected CV-1 cells, suggesting the presence of a nucleosomal structure. Transcription of NPCs was analyzed in vitro by using Escherichia coli DNA-dependent RNA polymerase. Both histone H1 and the fraction consisting of all five histones inhibited transcription of SV40 DNA by about 90 to 95%. The fraction consisting of four histones lacking H1 reduced the transcription by 30 to 35%, to a level similar to that of transcription with native NPCs. Transcription was inhibited regardless of whether the origin of histones was permissive or nonpermissive cells. Gel electrophoretic patterns of RNA products transcribed from SV40 DNA and reconstituted and native NPCs showed several identical peaks between 13S and 28S. The patterns were identical whether NPCs reconstituted with H1 alone, all five histones, or four histones lacking H1 were used.

DNA, Viral↗

PubMed1978

Presence of RNA in the nucleoprotein complex spontaneously released by human lymphocytes and frog auricles in culture.

Cell systems as different as normal human blood lymphocytes and frog auricles release spontaneously a nucleoprotein complex in their culture medium. This release seems to be an active mechanism that is unrelated to cell death. The presence of RNA in this complex is demonstrated. The amount of extracellular RNA is regulated by the same homeostatic mechanism that has previously been shown to govern DNA release in the same cell systems. This extracellular RNA is linked by hydrogen bonds to the extracellular DNA and cannot be extracted by a usual phenol procedure, due perhaps to the presence of a glycoprotein. Further purifications by chloroform, sodium perchlorate, and hydroxyapatite are necessary to obtain an RNA molecule that is acid precipitable, RNase and KOH sensitive, and orcinol positive. The extracellular RNA sediments between 2.5 and 4S and is not a transfer RNA. It is more highly methylated than the 28S, 18S, and 4 to 5S cellular RNA. It activates DNA synthesis in vitro.

Animals↗

PubMed1990

A novel nucleoprotein complex at a replication origin.

The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core. The interaction occurs through the DNA minor groove. The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.

Bacillus subtilis↗

PubMed1996

Chromatin nucleoprotein complexes containing tightly bound c-abl, p53 and bcl-2 gene sequences: correlation with progression of chronic myelogenous leukemia.

We previously developed a technique to isolate subchromatin nucleoprotein complexes (NPC) that contain tightly bound genes and enzymatic activities. NPC fractions (NPCF) were prepared by directly treating isolated nuclei with MspI to generate six NPCF (S1, M1, S2, M2, 0.1K and R). The NPCF have been used to predict the potential efficacy of interferon-alpha (IFN-alpha) treatment in patients with chronic myelogenous leukemia (CML) [Nicolson et al., Gene 159 (1995) 105-111]. Here the NPCF were probed for the presence of tightly bound c-abl, p53 and bcl-2 genes. We found that the NPCF isolated from the nuclei of leukocytes of normal individuals rarely contained detectable quantities of tightly-bound c-abl, p53 or blc-2 genes or gene sequences, whereas in CML nuclei these genes were often found in tight association with multiple NPCF. Examination of NPCF isolated from the leukocyte nuclei from patients with highly progressive CML for the presence of the three genes revealed that more NPCF contained the three tightly-bound genes than leukocyte NPCF from patients with stable or less progressed CML. These data suggest that as CML progresses to more malignant states, oncogenes, suppressor genes and apoptosis-associated genes become tightly associated with NPCF.

Adult↗

PubMed1995

Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination.

Regulation of transcription involves the assembly of multiprotein complexes at enhancers and promoters. Interactions between adjacent and non-adjacent DNA-binding proteins can augment the specificity and stability of multi-component nucleoprotein complexes. Recently, several proteins have been identified that can function as 'architectural' elements in the assembly of higher-order nucleoprotein structures reminiscent of those involved in site-specific recombination in prokaryotes.

Animals↗

PubMed1996

Genetic analysis of the bacteriophage lambda attL nucleoprotein complex.

Site-specific recombination in bacteriophage lambda involves interactions among proteins required for integration and excision of DNA molecules. We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF). Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL. IHF, in addition to Int, is required for efficient Int-core binding. We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes. Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes. Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively. In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the lambda arm-type sites. We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the lambda core sites.

Bacterial Proteins↗

PubMed1991

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.

Bacterial Proteins↗

PubMed1994

Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins.

Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination. HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity. We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase. The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA. We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure. We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome. HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions. These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts. Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA. While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies. This probably reflects subtle structural differences between the assembled complexes.

Allosteric Regulation↗

PubMed2005

Viral RNA is required for the association of APOBEC3G with human immunodeficiency virus type 1 nucleoprotein complexes.

APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5'-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.

5' Untranslated Regions↗

PubMed1980

Ready separation of proteins from nucleoprotein complexes by reversible modification of lysine residues.

Modification of proteins with citraconic anhydride altered the electrostatic relationship between cationic epsilon-NH3+ groups of lysine residues of proteins and anionic phosphate groups of nucleic acids, thereby destabilizing the nucleoprotein complex. This procedure facilitated the separation of proteins from nucleic acids at pH4-4.2. The modifying groups were then deacylated from the proteins under acidic conditions (pH3-6) at 30 degrees C.

Citraconic Anhydrides↗

PubMed1980

Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions.

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-N-N'-tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP(1) and VP(2) in addition to the proteins found associated with the viral minichromosome, i.e., VP(3) and histones H(2)A, H(2)B, H(3), and H(4). Our results suggest that the association of VP(1), VP(2), or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the "early" SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP(1) or VP(2) or both enhances the transcriptional activity and suggest that these "late" viral proteins may play a role in the regulation of expression of the SV40 genome.

Cell-Free System↗

PubMed1993

Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication.

Initiation of bacteriophage phi 29 DNA replication is activated by protein p6, a viral double-stranded DNA-binding protein that forms a nucleoprotein complex at the viral replication origins. This complex consists of a DNA right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp). In this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a phi 29 replication origin. DNase I footprinting shows that protein p6 binds to the concatemer in a similar way to the phi 29 DNA replication origins but with higher affinity, indicating that the 24 bp sequence is a recognition signal for protein p6. Furthermore, the concatemer was cloned in a plasmid and, by electron microscopy, it was shown to be the highest-affinity protein p6 binding region present in the plasmid. Based on these observations, the linking number change restrained by protein p6 has been measured in a series of plasmids containing concatemers with different numbers of 24 bp repeats; from the values obtained the linking number change restrained by a single protein p6 dimer has been estimated (delta Lkd = 0.1). In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts 4.2-fold the length of naked DNA. These data, together with the previously known value of the surface-related DNA helical repeat in the complex (12 bp), completely define the superhelical path of the DNA in the complex: one superhelical turn approximately involves 63 bp and 2.6 protein p6 dimers, and the DNA superhelix has a diameter of 6.6 nm and a slope of 14 degrees. The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11.5 bp/turn) and strongly bent (66 degrees/12 bp). These DNA conformational changes might contribute to the activation of phi 29 DNA initiation of replication by protein p6.

Bacillus Phages↗

PubMed2003

Visible light irradiation of ethidium bromide-stained interphase nuclei causes DNA-protein linking and structural stabilization of nucleoprotein complexes.

Fixation of DNA and proteins in the isolated rat hepatocyte nuclei stained with ethidium bromide and irradiated with visible light was analyzed in this study. It was shown that irradiation results in the following modifications of higher-level nucleoprotein complexes of interphase chromatin: (1) the complexes acquire resistance to decondensing treatments, which may be indicative of the formation of links between proteins or proteins and DNA in the chromatin; (2) the linking rate for both DNA and proteins is dose dependent; (3) the irradiation induces intermolecular link formation between DNA molecules, which brings about an increase in the average molecular weight of DNA fragments; (4) some modifications (dimerization, etc.) of histones and nonhistone proteins occur; and (5) histone proteins are not effectively cross-linked to DNA. The structural stabilization of interphase chromatin is possibly mediated by free radical-based mechanisms, whereas disulfide bonds seem to play no significant role in the cross-linking.

Animals↗

PubMed1999

Cooperative assembly of androgen receptor into a nucleoprotein complex that regulates the prostate-specific antigen enhancer.

Prostate cancer is characterized by elevated serum levels of prostate-specific antigen (PSA). PSA gene expression is controlled by an androgen-responsive transcriptional enhancer. Our study suggests that formation of a nucleoprotein complex, encompassing 170 base pairs of enhancer DNA, mediates androgen-responsive PSA enhancer activity. The complex is assembled by cooperative binding of androgen receptor to at least four tandem, nonconsensus androgen response elements (AREs). Systematic mutagenesis of the AREs demonstrated that they act synergistically to stimulate androgen receptor-responsive gene expression. We discuss a mechanism whereby a combination of high androgen receptor levels in the prostate and low affinity AREs contribute to the cell type specificity and activity of the enhancer.

Gene Expression Regulation↗

PubMed2000

Barrier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex.

Barrier-to-autointegration factor (BAF) is a highly conserved cellular protein that was identified by its activity in protecting retroviral DNA against autointegration. We show that BAF has the property of bridging double-stranded DNA in a highly ordered nucleoprotein complex. Whereas BAF protein alone is a dimer in solution, upon binding DNA, BAF forms a dodecamer with DNA bound at multiple discrete sites in the complex. The interactions between BAF and DNA are entirely nonspecific with respect to DNA sequence. The dual interaction of BAF with DNA and LAP2, a protein associated with the nuclear lamina, suggests a role for LAP2 in chromosome organization. Consistent with this idea, RNA interference experiments with Caenorhabditis elegans reveal a defect in mitosis.

Animals↗

PubMed2003

A nucleoprotein complex containing Sp1, C/EBP beta, and HMGI-Y controls human insulin receptor gene transcription.

HMGI-Y is an architectural transcription factor that regulates gene expression in vivo by controlling the formation of stereospecific multiprotein complexes on the AT-rich regions of certain gene promoters. Recently, we demonstrated that HMGI-Y is required for proper transcription of the insulin receptor (IR) gene. Here we provide evidence that transcriptional activation of the human IR promoter requires the assembly of a transcriptionally active multiprotein-DNA complex which includes, in addition to HMGI-Y, the ubiquitously expressed transcription factor Sp1 and the CCAAT-enhancer binding protein beta (C/EBP beta). Functional integrity of this nucleoprotein complex is required for full transactivation of the IR gene by Sp1 and C/EBP beta in cells readily expressing IRs. We show that HMGI-Y physically interacts with Sp1 and C/EBP beta and facilitates the binding of both factors to the IR promoter in vitro. Furthermore, HMGI-Y is needed for transcriptional synergism between these factors in vivo. Repression of HMGI-Y function adversely affects both Sp1- and C/EBP beta-induced transactivation of the IR promoter. Together, these findings demonstrate that HMGI-Y plays significant molecular roles in the transcriptional activities of these factors in the context of the IR gene and provide concordant support for the hypothesis that, in affected individuals, a putative defect in these nuclear proteins may cause decreased IR expression with subsequent impairment of insulin signaling and action.

3T3 Cells↗

PubMed1992

Electron spectroscopic imaging of chromatin and other nucleoprotein complexes.

Electron spectroscopic imaging (ESI) is a microanalytical technique which is being used to determine elemental distributions at the resolution limit of the electron microscope. Detection and mapping of phosphorus by energy filtered imaging makes it possible to determine the organization of the nucleic acid component in nucleoprotein complexes, because phosphorus is present at much higher levels in nucleic acids than in the associating proteins. ESI has provided a method for approaching numerous questions related to chromatin structure at the level of the specific protein-DNA interactions, at the nucleosome level and at higher organizational levels of chromosome structure.

Animals↗

PubMedDate not supplied

DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes.

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.

Animals↗