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PubMed1983

Simian virus 40 chromatin interaction with the capsid proteins.

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.

Capsid↗

PubMed1978

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

Adenoviruses, Human↗

PubMed1998

Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G2 phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in Xenopus laevis oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr's functions may take place at the NPC.

Biological Transport↗

PubMed2000

Preferential binding of fd gene 5 protein to tetraplex nucleic acid structures.

The gene 5 protein of filamentous bacteriophage fd is a single-stranded DNA-binding protein that binds non-specifically to all single-stranded nucleic acid sequences, but in addition is capable of specific binding to the sequence d(GT(5)G(4)CT(4)C) and the RNA equivalent r(GU(5)G(4)CU(4)C), the latter interaction being important for translational repression. We show that this sequence preference arises from the formation of a tetraplex structure held together by a central block of G-quartets, the structure of which persists in the complex with gene 5 protein. Binding of gene 5 protein to the tetraplex leads to formation of a approximately 170 kDa nucleoprotein complex consisting of four oligonucleotide strands and eight gene 5 protein dimers, with a radius of gyration of 45 A and an overall maximum dimension of 120-130 A. A model of the complex is presented that is consistent with the data obtained. It is proposed that the G-quartet may act as a nucleation site for binding gene 5 protein to adjacent single-stranded regions, suggesting a novel mechanism for translational repression.

Chromatography, Gel↗

PubMed1992

KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer.

We have constructed a RP4 KorB overproducing strain and purified the protein to near homogeneity. KorB is a DNA binding protein recognizing defined palindromic 13-bp sequences (TTTAGCSGCTAAA). Inverted sequence repetitions of this type, designated OB, are present on RP4 12 times. OB-sequences are localized in replication and maintenance regions as well as in the regions Tra1 and Tra2 essential for conjugative transfer. All sites found in Tra regions by computer search act as targets for specific binding of KorB protein. KorB-DNA complexes were detected by DNA fragment retardation assay using polyacrylamide gels. The 13-bp symmetric arrangement of the consensus OB-sequence constitutes the core for binding KorB protein since any truncation of this sequence prevents complex assembly or leads to a considerable destabilization of the KorB-DNA complexes. A hydroxyl radical footprint analysis demonstrated complex formation of KorB with the OB-sequence directly and suggests the presence of an unusual DNA structure within the nucleoprotein complex.

Amino Acid Sequence↗

PubMed1998

The spacing between binding sites controls the mode of cooperative DNA-protein interactions: implications for evolution of regulatory circuitry.

The CI repressors of lambdoid phages bind cooperatively to adjacent binding sites. Although these binding sites are found at complex operators containing three binding sites, cooperative binding occurs only between dimers bound at two of the sites, a mode of binding termed pairwise cooperativity. At the level of regulation, pairwise cooperativity allows the proper operation of the genetic switch. At the mechanistic level, it has been proposed to result from steric distortion of the complex, such that a protein dimer bound to a central site cannot contact both flanking dimers because it "leans" towards one of the two sites. We have tested this model using the CI repressor of phage HK022. Previous work suggested that reducing the spacing between adjacent operators might allow cooperative interaction among three dimers, a mode of cooperativity we term extended. Using a set of synthetic templates, we have shown that reducing the spacing allows extended cooperativity among three binding sites. Accordingly, the mode of cooperativity changes qualitatively in response to changes in the inter-site spacing. That is, the change in spacing has major functional consequences for the properties of the complex. We suggest that such changes can play important roles in the evolution of gene regulatory circuitry and of other processes involving nucleoprotein complexes.

Bacteriophage lambda↗

PubMed1984

Characterization and properties of a 5S-RNA-protein complex released from heated 60S ribosomal subunits.

When rat liver 60S ribosomal subunits were heated in phosphate buffer in the presence of MgCl2, 5S RNA was released in the form of a nucleoprotein complex (RNPH), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex: the acidic phosphoproteins P1-P2 and, as weaker spots, L3-L4, L6-L7 and L22. Most of these proteins were also found, but only as traces, in the RNPEDTA used as a control. RNPH was able to associate with 40S subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl-transferase center.

Animals↗

PubMed1977

Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class.

Adenoviridae↗

PubMed1979

[Conformational changes in the heat-denatured nucleotide complex of human lymphocytes on subsequent cooling].

The microfluorometric technique allowed an assay of the chromatin state from binding of acridine orange in studies on the kinetics of conformational changes in the nucleoprotein complex (NP) of human peripheral blood lymphocytes. Extraction of F1 histone fraction with 0.01 N HCl (pH 2) substantially altered (decelerated) conformational changes in the NP complex. Extraction of F1, F2 and F3 histone fractions with 0.25N HCl (pH 0.6) led to a more pronounced deceleration of the kinetics of conformational changes. The latter ones were decelerated as well when the process was run in a more viscous medium (e. g. in a 70% glycerin solution). No conformational changes in the NP complex deproteinized with 0.25N HCl were revealed in a vicsous medium. In such a case the NP complex showed low F530 values and elevated values of the alpha coefficient that seems likely to suggest the nonreversible denaturation of the NP complex.

Cell Nucleus↗

PubMed2000

Inhibition of nuclear factor-kappab activation in mouse macrophages and the RAW 264.7 cell line by a synthetic adenyl carbocyclic nucleoside.

Adenyl carbocyclic nucleosides have potent anti-inflammatory effects on a number of cell types. Notable in this regard is their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) by mouse macrophages that have been activated with bacterial lipopolysaccharide (LPS). Because the transcriptional activation of the mouse TNF-alpha gene is highly dependent on kappaB enhancers, the present study determined whether the synthetic carbocyclic nucleoside 9-[(1S,3R)-cis-cyclopentan-3-ol]adenine (cPA) inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in these cells. Stimulation of either mouse peritoneal macrophages or RAW 264. 7 macrophage-like cells with LPS led to the appearance of four distinct kappaB-binding nucleoprotein complexes detected by gel mobility shift assays. Cells treated with 100 microM cPA showed significantly reduced levels of NF-kappaB activation as evidenced by measurements of nucleoprotein kappaB-binding activity and diminished kappaB-dependent transcriptional activation. However, both the LPS-induced degradation of the cytoplasmic NF-kappaB inhibitor IkappaBalpha and the nuclear translocation of the NF-kappaB p50, p65, and c-Rel peptides were unaffected by treatment of the cells with the nucleoside. These findings suggest that certain adenyl carbocyclic nucleosides inhibit the activation of NF-kappaB/Rel complexes by a novel mechanism that results in an inhibition of their DNA-binding activities, without blocking their dissociation from IkappaBalpha or their nuclear translocation.

Adenine↗

PubMed2007

New insights into the nucleophosmin/nucleoplasmin family of nuclear chaperones.

Basic proteins and nucleic acids are assembled into complexes in a reaction that must be facilitated by nuclear chaperones in order to prevent protein aggregation and formation of non-specific nucleoprotein complexes. The nucleophosmin/nucleoplasmin (NPM) family of chaperones [NPM1 (nucleophosmin), NPM2 (nucleoplasmin) and NPM3] have diverse functions in the cell and are ubiquitously represented throughout the animal kingdom. The importance of this family in cellular processes such as chromatin remodeling, genome stability, ribosome biogenesis, DNA duplication and transcriptional regulation has led to the rapid growth of information available on their structure and function. The present review covers different aspects related to the structure, evolution and function of the NPM family. Emphasis is placed on the long-term evolutionary mechanisms leading to the functional diversification of the family members, their role as chaperones (particularly as it pertains to their ability to aid in the reprogramming of chromatin), and the importance of NPM2 as an essential component of the amphibian chromatin remodeling machinery during fertilization and early embryonic development.

Animals↗

PubMed2004

Loss of net as repressor leads to constitutive increased c-fos transcription in cervical cancer cells.

We have investigated the expression of c-fos in cervical carcinoma cells and in somatic cell hybrids derived therefrom. In malignant cells, c-fos was constitutively expressed even after serum starvation. Dissection of the c-fos promoter showed that expression was mainly controlled by the SRE motif, which was active in malignant cells, but repressed in their non-malignant counterparts. Constitutive SRE activity was not mediated by sustained mitogen-activated protein kinase activity but because of inefficient expression of the ternary complex factor Net, which was either very low or even barely discernible. Chromatin immunoprecipitation assays revealed that Net directly binds to the SRE nucleoprotein complex in non-tumorigenic cells, but not in malignant segregants. Small interfering RNA targeted against Net resulted in enhanced c-fos transcription, clearly illustrating its repressor function. Conversely, stable ectopic expression of Net in malignant cells negatively regulated endogenous c-fos, resulting in a disappearance of the c-Fos protein from the AP-1 transcription complex. These data indicate that loss of Net and constitutive c-fos expression appear to be a key event in the transformation of cervical cancer cells.

Cell Transformation, Neoplastic↗

PubMed1996

Molecular determinants of mammmalian sex.

Mammalian male sex determination is controlled by a complex hierarchy of gene regulatory proteins and hormones, which promote male gonadal development and regression of the female primordia. At the core of this pathway lies the SRY protein, the master developmental switch for testicular differentiation and hence, the male sex. The three-dimensional structure of the SRY-DNA complex suggests a model of developmental gene regulation in which proteins that alter DNA structure and promote the assembly of higher-order nucleoprotein complexes play an essential role in the timing of cell specialization events.

Animals↗

PubMed1996

Histone-like protein HU as a specific transcriptional regulator: co-factor role in repression of gal transcription by GAL repressor.

BACKGROUND: Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters. Coordinated repression of the two promoters requires GalR binding to both operators. In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivo. RESULTS: We have purified a protein that restored the expected GalR-mediated repression for the in vitro system and have identified this factor to be the bacterial histone-like protein HU. In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, D-galactose. The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation. CONCLUSION: We propose that HU, acting as a co-factor, brings about the GalR-mediated repression by forming a distinct nucleoprotein complex of higher order structure. Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex.

Bacterial Proteins↗

PubMedDate not supplied

[Cooperative interaction of serum albumin with quaternized poly-4-vinyl pyridine and structure of the complexes].

Interaction of bovine serum albumin (BSA) with quaternized poly-4-vinyl pyridine (PE) in aqueous solutions at pH 7 was studied. It was shown that in a wide range of the ratios of the components (nBSA/nPE) soluble stable cooperative complexes were formed. At the same time a certain critical content of the protein exists at which the system loses its homogeneity. Complex formation is not accompanied by protein denaturation. At smaller nBSA/nPE ratios non-homogeneous distribution of protein globulas among polyelectrolite macromolecules was found; this corresponded to the "all or none" principle. Using ultracentrifugation technique viscosimetric measurements and electron microscopy it was shown that the soluble complexes exist in the form of rode-like particles consisting of protein globules stabilized by polycation chains. Such particle can be considered as a model of nucleoprotein complex. At certain crytical nBSA/nPE rations the rod-like particles aggregate with additional number of BSA-molecules and form more complicate soluble and insoluble cooperative complexes. Possible structural models of the complexes described were suggested and the thermodinamic and kinetic cryteria of their self-assembly were discussed.

Drug Stability↗

PubMed1983

Simian virus 40 morphogenetic pathway. An analysis of assembly-defective tsB201 DNA protein complexes.

Under restrictive conditions, the 220 S SV40 virions are not assembled in tsB201-infected cells. Instead, a new class of SV40 DNA-containing particles is isolated in addition to the 75 S chromatin. This new class of nucleoprotein complex sediments heterogeneously between 100 to 160 S with a peak at 130 S. Under an electron microscope, these complexes appear predominantly as SV40 chromatin associated with a shell-like protein cluster. These structures resemble the wild type assembly intermediates previously observed by Coca-Prados and Hsu (Coca-Prados, N., and Hsu, M.-T. (1979) J. Virol. 31, 199-208). Like the wild type assembly intermediates, the tsB201 DNA-protein complexes are unstable in high salt. In CsCl, they yield a protein species with a density characteristic of empty shells. In 1 M NaCl, they release heterogeneous 55-110 S protein polymers which consist of the capsid proteins VP1, VP2, and VP3. Our results indicate that the tsB201 nucleoproteins consist of capsid proteins, with varying extents of polymerization, held to chromatin by electrostatic bonds. The accumulation of these nucleoproteins is consistent with a simian virus 40 morphogenetic pathway wherein the capsid proteins are added gradually to the 75 S chromatin.

Cell Transformation, Neoplastic↗

PubMed1994

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

Adenosine Triphosphatases↗

PubMed1997

Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression.

In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required. The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.

Base Sequence↗