The arrangement of nucleosomes in nucleoprotein complexes from polyoma virus and SV40.
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Base Sequence↗
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Results for “nucleoprotein complex”
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Architectural elements in nucleoprotein complexes.
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Journal Article↗
High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex.
We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.
Adenoviruses, Human↗
DNA looping by Saccharomyces cerevisiae high mobility group proteins NHP6A/B. Consequences for nucleoprotein complex assembly and chromatin condensation.
The formation of higher order protein.DNA structures often requires bending of DNA strands between specific sites, a process that can be facilitated by the action of nonspecific DNA-binding proteins which serve as assembly factors. A model for this activity is the formation of the invertasome, an intermediate structure created in the Hin-mediated site-specific DNA inversion reaction, which is stimulated by the prokaryotic nucleoid-associated protein HU. Previously, we have shown that the mammalian HMG1/2 proteins substitute for HU in this system and display efficient DNA wrapping activity in vitro. In the present work, we isolate the primary sources of assembly factor activity in Saccharomyces cerevisiae, as measured by the ability to stimulate invertasome formation, and show that these are the previously identified NHP6A/B proteins. NHP6A/B have comparable or greater activity in DNA binding, bending, and supercoiling with respect to HU and HMG1 and appear to form more stable protein.DNA complexes. In addition, expression of NHP6A in mutant Escherichia coli cells lacking HU and Fis restores normal morphological appearance to these cells, specifically in nucleoid condensation and segregation. From these data we predict diverse architectural roles for NHP6A/B in manipulating chromosome structure and promoting the assembly of multicomponent protein.DNA complexes.
Base Sequence↗
Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors.
The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. Id proteins are thought to inhibit differentiation mainly through interaction with other HLH proteins and by blocking their DNA-binding activity. Members of the ternary complex factor (TCF) subfamily of ETS-domain proteins have key functions in regulating immediate-early gene expression in response to mitogenic stimulation. TCFs form DNA-bound complexes with the serum response factor (SRF) and are direct targets of MAP kinase (MAPK) signal transduction cascades. In this study we demonstrate functional interactions between Id proteins and TCFs. Ids bind to the ETS DNA-binding domain and disrupt the formation of DNA-bound complexes between TCFs and SRF on the c-fos serum response element (SRE). Inhibition occurs by disrupting protein-DNA interactions with the TCF component of this complex. In vivo, the Id proteins cause down-regulation of the transcriptional activity mediated by the TCFs and thereby block MAPK signalling to SREs. Therefore, our results demonstrate a novel facet of Id function in the coordination of mitogenic signalling and cell cycle entry.
3T3 Cells↗
DNA bending in the ternary nucleoprotein complex at the c-fos promoter.
Transcriptional induction of the c-fos proto-oncogene in response to serum growth factors is mediated in part by a ternary complex that forms on the serum response element (SRE) within its promoter. This complex consists of Elk-1, serum response factor (SRF) and the SRE. Elk-1 is phosphorylated by MAP kinase, which correlates with the induction of c-fos transcription. In this study we have investigated the protein-induced DNA bending which occurs during the formation and post-translational modification of the ternary complex that forms at the c-fos SRE. Circular permutation analysis demonstrates that the minimal DNA-binding domain of SRF, which contains the MADS box, is sufficient to induce flexibility into the centre of its binding site within the SRE. Phasing analysis indicates that at least part of this flexibility results in the production of a directional bend towards the minor groove. The isolated ETS domains from Elk-1 and SAP-1 induce neither DNA bending nor increased DNA flexibility. Formation of ternary complexes by binding of Elk-1 to the binary SRF:SRE complex results in a change in the flexibility of the SRE. Phosphorylation of Elk-1 by MAP kinase (p42/ERK2) induces further minor changes in this DNA flexibility. However, phasing analysis reveals that the recruitment of Elk-1 to form the ternary complex affects the SRF-induced directional DNA bend in the SRE. The potential roles of DNA bending at the c-fos SRE are discussed.
Base Sequence↗
DNA polymerases and a single-strand-specific DNA-binding protein associated with simian virus 40 nucleoprotein complexes.
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DNA Helicases↗
Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.
The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.
Capsid↗
Intracellular forms of simian virus 40 nucleoprotein complexes. V. Enrichment for "active" simian virus 40 chromatin by differential precipitation with Mg2+.
A fraction (approximately 20%) of 70S simian virus (SV40) chromatin isolated from a sucrose gradient-purified extract of SV40-infected CV-1 cells was found to be precipitated by incubation with a buffer containing 10 mM Mg2+ at 37 degrees C. The fraction containing the Mg2+-insoluble SV40 chromatin was found also to be enriched for the following: (i) DNA replication intermediates as analyzed by short pulse with [3H]thymidine; (ii) transcription complexes as assayed by in vitro synthesis of SV40-specific RNA; (iii) newly synthesized T antigen; (iv) phosphorylated T antigen as analyzed by both in vitro and in vivo phosphorylation studies; and (v) ATP-binding activity of T antigen as assayed by affinity labeling with oxidized [alpha-32P]ATP.
Adenosine Triphosphate↗
ND10 components relocate to sites associated with herpes simplex virus type 1 nucleoprotein complexes during virus infection.
Infections with DNA viruses commonly result in the association of viral genomes and replication compartments with cellular nuclear substructures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. While there is evidence that viral genomes can associate with preexisting ND10, we demonstrate in this study by live-cell microscopy that structures resembling ND10 form de novo and in association with viral genome complexes during the initial stages of herpes simplex virus type 1 (HSV-1) infection. Consistent with previous studies, we found that the major ND10 proteins PML, Sp100, and hDaxx are exchanged very rapidly between ND10 foci and the surrounding nucleoplasm in live cells. The dynamic nature of the individual protein molecule components of ND10 provides a mechanism by which ND10 proteins can be recruited to novel sites during virus infection. These observations explain why the genomes and replication compartments of DNA viruses that replicate in the cell nucleus are so commonly found in association with ND10. These findings are discussed with reference to the nature, location, and potential number of HSV-1 prereplication compartments and to the dynamic aspects of HSV-1 genomes and viral products during the early stages of lytic infection.
Animals↗
CCAAT-binding factor NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that spans 250 base pairs: the invariant chain promoter as a model.
The events that lead to promoter accessibility within chromatin are not completely understood. The invariant chain (Ii) promoter was used as a model to determine the contribution of different DNA-binding factors in establishing occupancy of a complex promoter. Gamma interferon induction of the Ii promoter requires the cooperation of multiple cis elements including distal S, X, and Y/CCAAT elements along with proximal GC and Y/CCAAT elements. The heteromeric transcription factor NF-Y binds to both Y/CCAAT elements. Genomic footprinting was used to analyze in vivo protein-DNA contacts for integrated Ii promoters bearing mutations in each element. The results reveal a hierarchy of transcription factor loading with NF-Y binding to the distal Y/CCAAT element being required for establishing protein-DNA interactions over the entire 250 bp analyzed. Mutation of the X box disrupts binding primarily at the adjacent Y/CCAAT element along with a lesser effect on GC box binding. Importantly, this finding is verified with a cell line which lacks a functional X-box-binding factor, RFX, providing physiological validity for the strategy described here. Mutation of both the S element and the GC box results in either no or little effect on transcription factor binding. However, mutation of the proximal Y/CCAAT element disrupts binding to the adjacent GC box and partially reduces binding in the distal S/X/Y domain. The crucial role for NF-Y in establishing promoter occupancy may be related to its histone fold motif, the essential component for assembling nucleosome-like structures.
Antigens, Differentiation, B-Lymphocyte↗
Anatomy of the primase-alpha DNA polymerase reaction accomplished by nucleoprotein complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenyl deoxyguanosine-5'-triphosphate.
Activities of the primase (Pr)-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring reaction systems represented by special NP complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (J. Riman, A. Sulová and K. Horská, Acta virol. 39, 149-159 (1995); J. Ríman and A. Sulová, Acta virol. 41, 181-192 (1997)) were studied in the absence and presence of carbonyldiphosphonate (COMDP), mimosine (MIMO), to it related ciclopirox olamine (CPX) and butylphenyl deoxyguanosine-5'-triphosphate (BuPdGTP). Reaction products radioactively labeled for RNA and DNA and synthesized with the common four ribonucleoside triphosphates (rNTPs) or rNTPs and deoxyribonucleoside triphosphates (dNTPs) in the reaction medium, were analyzed by polyacrylamide gel electrophoresis (PAGE) at denaturing conditions. It was shown that COMDP strongly activates the Pr and uncouples its activity from that of alpha DNA pol with accumulation of initiator (i) RNAs of the basic length. This phenomenon is not affected by BuPdGTP. MIMO, in contrast, stimulates both pol activities of this enzyme complex and preserves their mutual coupling. The effects of COMDP, MIMO and CPX seem to be modulated by concentration of the ambient dNTPs. Addition of dNTPs to rNTPs makes the effects of COMDP and MIMO mutually exclusive, suggesting that both these agents, though chemically quite different, are competing for one active site responsible for coupling these both pol activities into the one Pr-alpha DNA pol reaction.
Avian Myeloblastosis Virus↗
Pyrimidine regulation of the Escherichia coli and Salmonella typhimurium carAB operons: CarP and integration host factor (IHF) modulate the methylation status of a GATC site present in the control region.
By measuring the protection against Dam methylase modification of a GATC sequence located 106 bp upstream of the startpoint of promoter P1 in the control region of the carAB operon (encoding carbamoylphosphate synthetase) we have obtained evidence for a direct correlation between the degree of in vivo occupancy of a specific regulatory target site and the repressibility of the P1 promoter by pyrimidine residues. A high uridine nucleotide pool as well as binding of the carP (alias xerB/pepA) gene product and of the integration host factor (IHF) to the carAB control region are prerequisites to observe this in vivo protection. Purified CarP binds in vitro to the carAB control region and protects against DNase I two approximately 25 bp long stretches, one of which is located just downstream of the GATC sequence. Mutations in this site strongly impair the pyrimidine regulation of the P1 promoter and the interference with Dam methylase modification. These processes are also strongly impaired in the absence of integration host factor and in mutants affected in the IHF site located some 200 bp upstream of this Dam methylase modification site. IHF therefore exerts at least part of its antagonistic effects on P1, i.e. increased expression in minimal medium but increased repression in the presence of pyrimidine residues, indirectly by influencing the formation or the stability of a particular protein-DNA complex. Furthermore, we demonstrate that the distance separating the IHF and Dam methylase target sites is crucial for the in vivo protection and for pyrimidine-mediated regulation of the promoter expression. Mutations altering this distance result in severe reductions of the degree of in vivo protection and, concomitantly, of the repressibility by pyrimidine residues of promoter P1 activity in a way indicative of the formation of a complex nucleoprotein structure. Since neither IHF nor CarP require pyrimidine residues to bind to the carAB control region, at least not in vitro, it is tempting to suggest that IHF and CarP-induced bending and looping provide changes in DNA topology that are required for assembling a specific pyrimidine-dependent nucleoprotein complex that modulates P1 activity.
Adenine↗
FIS activates sequential steps during transcription initiation at a stable RNA promoter.
FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.
Carrier Proteins↗
DNA trajectory in the Gal repressosome.
The Gal repressosome is a higher-order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli. During the repressosome assembly, a DNA loop is formed by the interaction of two GalR dimers, bound to two spatially separated operators, OE and OI, flanking the gal promoters. Structure-based genetic analysis indicated that GalR homodimers interact directly and form a V-shaped stacked tetramer in repressosome, further stabilized by HU binding to an architecturally critical position on the DNA. In this scheme of GalR tetramerization, the alignment of the operators in the DNA loop could be in either parallel (PL) or antiparallel (AL) mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE- and OI-bound GalR dimers, it is possible to have four different DNA trajectories in the repressosome. Feasibilities of these trajectories were tested by in vitro transcription repression assays, first by isolating GalR mutants with altered operator specificity and then by constructing four different potential loops with mutant GalR heterodimers bound to specifically designed hybrid operators in such a way as to give rise to only one of the four putative trajectories. Results show that OE and OI adopt a mutual antiparallel orientation in an under-twisted DNA loop, consistent with the energetically optimal structural model. In this structure the center of the HU-binding site is located at the apex of the DNA loop. The approach reported here can be used to distinguish between otherwise indistinguishable DNA trajectories in complex nucleoprotein machines.
Bacterial Proteins↗
Intracellular DNA of the parvovirus minute virus of mice is organized in a minichromosome structure.
Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer. Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S. In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin. They were found to be composed of replicative forms of MVM DNA and cellular histones. After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified. These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively. We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.
Chromosomes↗
A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex.
Stable maintenance of the low-copy-number mini-F plasmid in Escherichia coli is dependent on a functional partition system. The sop partition region encodes proteins SopA and SopB and a cis-acting element sopC, which contains multiple sites to which SopB binds. We have found that SopB protein acting at sopC in vivo is associated with a marked effect on plasmid DNA supercoiling, which may reflect the formation of a wrapped nucleoprotein complex. In this study, we demonstrate that a functional partition complex can form with a single 43-bp SopB binding site. Our experiments suggest that SopB bound at a single site nucleates the binding of additional SopB protein and wrapping of adjacent DNA sequences, such that approximately equal numbers of supercoils are restrained regardless of the number of tandem sopC repeats present. It is likely that this unusual nucleoprotein complex allows interaction of the plasmid with the partition apparatus.
Base Sequence↗
Crystal structure of the rabies virus nucleoprotein-RNA complex.
Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.
Amino Acid Sequence↗