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At least 829 records · Page 46Linked to original sources
PubMed2000

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

Bacillus Phages↗
PubMed2001

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid.

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.

Amides↗
PubMedDate not supplied

Lambda vectors for stable cloned gene expression.

The bacteriophage lambda offers a unique opportunity concurrently to minimize segregational instability in recombinant systems by chromosomal integration of the cloned gene and to achieve high cloned gene expression during an abortive lytic phase. Lysis leads approximately to a 100-fold amplification of the cloned gene. Cell lysis in the lytic state is blocked by a specific mutation (Sam), allowing the cell to maintain its integrity, and lambda DNA packaging is blocked by other mutations (Wam, Eam) that keep cloned genes open to transcription. In the presence of these mutations, extremely high levels of cloned beta-galactosidase (more than 15% of total cell protein) have been obtained during abortive lysis from vectors found to be essentially 100% stable for over 75 generations in the lysogenic phase.

Bacteriophage lambda↗
PubMed2006

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry.

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.

Amino Acid Substitution↗
PubMed2005

Reversing histone methylation.

Histones package DNA, and post-translational modifications of histones can regulate access to DNA. Until recently, histone methylation-unlike all other histone modifications-was considered a permanent mark. The discovery of enzymes that reverse the methylation of lysines and arginines challenges our current thinking on the unique nature of histone methylation, and substantially increases the complexity of histone modification pathways.

Animals↗
PubMed2003

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.

Amino Acid Sequence↗
PubMed1995

Assembly of VP26 in herpes simplex virus-1 inferred from structures of wild-type and recombinant capsids.

The 1250 A diameter herpes simplex virus-1 (HSV-1) capsid shell consists of four major structural proteins, of which VP26 (approximately 12,000 M(r)) is the smallest. Using 400 kV electron cryomicroscopy and computer reconstruction, we have determined the three-dimensional structures of the wild-type capsid and a recombinant baculovirus-generated HSV-1 capsid which lacks VP26. Their difference map demonstrates the presence of VP26 hexamers attached to all the hexons in the wild-type capsid, and reveals that the VP26 molecule consists of a large and a small domain. Although both hexons and pentons are predominantly composed of VP5, VP26 is not present on the penton. Based on the interactions involving VP26 and the hexon subunits, we propose a mechanism for VP26 assembly which would account for its distribution. Possible roles of VP26 in capsid stability and DNA packaging are discussed.

Animals↗
PubMed2002

Minor proteins, mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid.

Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.

Bacteriophage PRD1↗
PubMed2007

Nutrient starvation promotes condensin loading to maintain rDNA stability.

Nutrient starvation or rapamycin treatment, through inhibition of target of rapamycin, causes condensation of ribosomal DNA (rDNA) array and nucleolar contraction in budding yeast. Here we report that under such conditions, condensin is rapidly relocated into the nucleolus and loaded to rDNA tandem repeats, which is required for rDNA condensation. Rpd3-dependent histone deacetylation is necessary and sufficient for condensin's relocalization and loading to rDNA array, suggesting that histone modification plays a regulatory role for condensin targeting. Rapamycin independently, yet coordinately, inhibits rDNA transcription and promotes condensin loading to rDNA array. Unexpectedly, we found that inhibition of rDNA transcription in the absence of condensin loading leads to rDNA instability. Our data suggest that enrichment of condensin prevents rDNA instability during nutrient starvation. Together, these observations unravel a novel role for condensin in the maintenance of regional genomic stability.

Adenosine Triphosphatases↗
PubMed2003

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus.

The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5'/3' ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems - exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5'/3' ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

Base Sequence↗
PubMed2001

Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death?

AIMS: Ultrastructural and molecular studies to support the hypothesis of programmed cell death in Helicobacter pylori were conducted. METHODS AND RESULTS: Evidence of programmed death in H. pylori is provided through electron microscopic detection and cytochemical labelling of electrondense bodies (EDB), containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic cells. This morphological evidence is also supported by DNA cleavage in homogeneous fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at 37 degrees C, with a maximum of 37.5% of EDB coccoid cells after 7 days. The non-permissive temperature of 4 degrees C anticipated this process, with 40% of EDB coccoid forms within 3 days, and it remained substantially unaffected during the observation time of 14 days. CONCLUSION: In these experiments, deprivation of nutrients and a non-permissive temperature acted as a powerful trigger for programmed cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Helicobacter pylori bacterial populations, under stressing stimuli, can respond with programmed cell suicide as a means of species preservation.

Apoptosis↗
PubMed2000

Oligomerization of the chromatin-structuring protein H-NS.

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Amino Acid Sequence↗
PubMed2003

RNA-modifying machines in archaea.

It has been known for nearly half a century that coding and non-coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non-coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA-containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small-non-coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

Amino Acid Sequence↗
PubMed2004

Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS.

For more complete characterization of DNA-predicted proteins (including their posttranslational modifications) a "top-down" approach using high-resolution tandem MS is forwarded here by its application to methanogens in both hypothesis-driven and discovery modes, with the latter dependent on new automation benchmarks for intact proteins. With proteins isolated from ribosomes and whole-cell lysates of Methanococcus jannaschii (approximately 1,800 genes) using a 2D protein fractionation method, 72 gene products were identified and characterized with 100% sequence coverage via automated fragmentation of intact protein ions in a custom quadrupole/Fourier transform hybrid mass spectrometer. Three incorrect start sites and two modifications were found, with one of each determined for MJ0556, a 20-kDa protein with an unknown methylation at approximately 50% occupancy in stationary phase cells. The separation approach combined with the quadrupole/Fourier transform hybrid mass spectrometer allowed targeted and efficient comparison of histones from M. jannaschii, Methanosarcina acetivorans (largest Archaeal genome, 5.8 Mb), and yeast. This finding revealed a striking difference in the posttranslational regulation of DNA packaging in Eukarya vs. the Archaea. This study illustrates a significant evolutionary step for the MS tools available for characterization of WT proteins from complex proteomes without proteolysis.

Amino Acid Sequence↗
PubMed2004

Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression.

Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1/L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML-/- cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML+ cells compared with the PML-/- cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML+ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.

Animals↗
PubMed2006

Formation of functional centromeric chromatin is specified epigenetically in Candida albicans.

In the pathogenic yeast Candida albicans, the 3-kb centromeric DNA regions (CEN) of each of the eight chromosomes have different and unique DNA sequences. The centromeric histone CaCse4p (CENP-A homolog) occurs only within these 3-kb CEN regions to form specialized centromeric chromatin. Centromere activity was maintained on small chromosome fragments derived in vivo by homologous recombination of a native chromosome with linear DNA fragments containing a telomere and a selectable marker. An in vivo derived 85-kb truncated chromosome containing the 3-kb CEN7 locus on 69 kb of chromosome 7 DNA was stably and autonomously maintained in mitosis, indicating that preexisting active CEN chromatin remains functional through many generations. This same 85-kb chromosome fragment, isolated as naked DNA (devoid of chromatin proteins) from C. albicans and reintroduced back into C. albicans cells by standard DNA transformation techniques, was unable to reform functional CEN chromatin and was mitotically unstable. Comparison of active and inactive CEN chromatin digested with micrococcal nuclease revealed that periodic nucleosome arrays are disrupted at active centromeres. Chromatin immunoprecipitation with antibodies against CaCse4p confirmed that CEN7 introduced into C. albicans cells as naked DNA did not recruit CaCse4p or induce its spread to a duplicate region only 7 kb away from active CEN7 chromatin. These results indicate that CaCse4p recruitment and centromere activation are epigenetically specified and maintained in C. albicans.

Base Pairing↗
PubMed1983

5-methylcytosine is localized in nucleosomes that contain histone H1.

We have developed a procedure for purifying highly specific polyclonal antibodies against 5-methylcytosine. These antibodies were used to probe the distribution of 5-methylcytosine among fractionated nucleosomes of mouse cell chromatin. Our results demonstrate that at least 80% of the 5-methylcytosine is localized in nucleosomes that contain histone H1. Native nucleosomes that lack histone H1 or possess high mobility group proteins package DNA that is 1.6- to 2.3-fold undermethylated. We suggest that the preferential association of methylated sequences with histone H1 has functional significance because DNA methylation has been linked to gene inactivation and histone H1 is known to promote chromatin condensation.

5-Methylcytosine↗
PubMed1993

Analysis of the mouse protamine 1 promoter in transgenic mice.

Protamines are small arginine-rich proteins that package DNA in spermatozoa. The mouse protamine 1 (Prm-1) gene is transcribed exclusively in post-meiotic spermatids. To identify elements in the Prm-1 promoter required for spermatid-specific transcription, we generated transgenic mice by microinjection of transgenes containing Prm-1 5' flanking sequences with 5' truncations or internal deletions of conserved sequences linked to a marked Prm-1 gene. We also tested Prm-1 promoter regions with a heterologous human growth hormone reporter gene. We conclude that a 113-bp region can direct spermatid-specific transcription and we have defined sequences within this region that are essential for proper function. These results will facilitate the isolation and characterization of transcription factors essential for post-meiotic gene expression.

Animals↗