Mechanics of chromatin template activation. Physical evidence for destabilization of nucleoproteins by polyanions.

Thermal denaturation profiles were used to quantitate the destabilizing activity of five classes of polyanions in an effort to explain previously reported differences in activity among apparently similar polymers. This physical assay for polyanion action on chromatin is free of the biochemical complications associated with template assay systems. Most polyanions tested caused a thermal destabilization of the normal nucleohistone complexes of rat thymus chromatin and exposed free or weakly complexed DNA. Polyphosphates dissociated chromatin even when their net charge was as low as 6, and activity per unit of weight increased with chain length only for polymers containing less than about 20 phosphate units; pyrophosphate was inactive. Two polymers which had no influence on chromatin had a low negative charge density. Some, such as polycytidlate and denatured DNA, possessed a high charge but caused only minor changes in the shape of denaturation profiles and exposed little or no free DNA. The highly active nucleic acids, ribosomal RNA and polyguanylate, were distinguishable from inactive nucleic acids by having more secondary structure and potentially less steric hindrance for anionic interaction with chromatin. There was evidence that some polyanions dissociate certain histones before others. Although data obtained show that some histones may be redistributed from chromatin onto added double-stranded DNA in a medium containing urea, this does not appear to interfere significantly with the interpretation of typical thermal denaturation profiles of chromatin.     

Structure and Dynamics of Double Helices in Solution: Modes of DNA Bending

Abstract

The long range structure of DNA restriction fragments has been analysed by electro-optical measurements. The overall rotation time constants observed in a low salt buffer with monovalent ions is shown to decrease upon addition of Mg²⁺ or spermine. Since the circular dichroism and also the limiting value of the linear dichroism remain almost constant under these conditions, the effect is attributed to a change of the long range structure. According to a weakly bending rod model, the persistence length decreases from about 600 Å in the absence of Mg²⁺ or spermine to about 350 Å in the presence of these ions. The persistence length measured in the presence of Mg²⁺ is almost independent of temperature in the range of 10 to 40 °C. The nature of DNA bending is analysed by measurements of bending amplitudes and time constants from dichroism decay curves. The observed absence of changes in the bending amplitudes upon addition of Mg²⁺ or spermine, even though addition induces changes of the persistence length by a factor of 2, is hardly consistent with simple thermal bending. The combined results, including the remarkably small temperature dependence of persistence length and bending amplitude, can be explained by the existence of two bending effects: inherent curvature of DNA dominates at low temperature, whereas thermal bending prevails at high temperature. Analysis of bending amplitudes from dichroism decay curves according to an arc model provides an approximate measure for the degree of bending in restriction fragments. The model is consistent with the observed chain length dependence of bending amplitudes and provides an approximate curvature corresponding to a radius of ab out400Å. Thus the curvature observed in restriction fragments is similar to that observed for high molecular DNA condensed into toroids by addition of ions like spermine.

Particularly strong bending of DNA is induced by [CO(NH₃)₆]³⁺, indicated by an apparent persistence length of 200 Å and an increased bending amplitude together with a reduced limit value of the linear dichroism. This effect is attributed to the high charge density of this ion and potential site binding.     

Terminase Subunits from the Pseudomonas-Phage E217

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ～58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.     

Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution.

gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

Structure of magnesium paracrystals of α-tropomyosin

The molecular packing of magnesium paracrystals of α-tropomyosin has been examined by electron microscopy. Previous work (Caspar et al., 1969) had shown that these structures are composed of antiparallel arrays of molecules and we have now studied the relative positions of the molecules by matching the banding patterns of paracrystals positively stained with uranyl acetate to the sequence of the molecule. The overlap between the C-termini of the molecules in the unit cell is 175 ± 2 residues and the overlap of the N-termini lies in the range 107 to 122 residues. In the long overlap region (between C-termini), and probably also in the short overlap region, the molecular packing is such that the periodic zones of negative charge present in the sequence (Stewart & McLachlan, 1975) lie opposite one another. We propose that magnesium bridges between opposing negative charges contribute strongly to the stability of the structure. We confirm earlier work (Stewart, 1975b) on the absolute orientation of the molecules in the paracrystal: the troponin binding site on tropomyosin is approximately 130 Å from the C-terminus, and Cys190 is within 10 to 15 Å units of the C-C dyad.     

The inorganic ion content of native aquatic bacteria.

In this study we have quantified the ionic content and volume of native aquatic, and two cultured bacteria, by X-ray microanalysis (XRMA) in the transmission electron microscope (TEM). The cellular concentrations of magnesium (means of 630 and 710 mM) were more than an order of a magnitude higher than the outside concentrations. The internal concentrations of sodium were on average 50-180 mM, and the [K+]/[Na+] ratios were in the range of 0.1-0.5; lowest for apparently nonactive bacteria. Magnesium and chloride probably act as the major components of cell turgor, since no other inorganic ions were present in comparable amounts. Our carbon and nitrogen measurements indicated that organic solutes are not likely to be present at significant concentrations. The estimated charge of inorganic ions (Na, Mg, P, Cl, K, and Ca) gave a positive net internal charge for most cells. However, in cultures of Vibrio natriegens, the high internal chloride concentration made the net inorganic charge negative in these cells. Our results suggest that growing marine bacterioplankton have an internal environment in which magnesium is the dominating cation. These results suggest that actively growing marine bacteria are physiologically adapted to high internal concentrations of both magnesium and chloride.     

Comparison of X-ray crystal structures of a tetradecamer sequence d(CCCGGGTACCCGGG)2 at 1.7 Å resolution

We present here a comparison of three different X-ray crystal structures of DNA tetradecamer sequence d(CCCGGGTACCCGGG)₂ all at about 1.7 Å resolution. The sequence was designed as an attempt to form a DNA four-way junction with A-type helical arms. However, in the presence of zinc, magnesium, and in the absence of any metal ion, it does not take up the junction structure, but forms an A-type double helix. This allowed us to study possible conformational changes in the double helix due to the presence of metal ions. Upon addition of the zinc ion, there is a change in the space group from P4₁2₁2 to P4₁. The overall conformation of the duplex remains the same. There are small changes in the interaction of the metal ions with the DNA. In the zinc-bound structure, there are two zinc ions that show direct interaction with the N7 atoms of terminal G₁₃ bases at either end of the molecule. There are small changes in the interhelical contacts. The consequence of these differences is to break some of the symmetry and change the space group.     

Ultraviolet radiation response to the physical properties of the reconstituted nucleohistone

The effect of UV irradiation on the reconstituted nucleohistone have been studied with reference to its (nucleohistone) changes in physical properties, after irradiation at different UV doses. The rate of fall of specific viscosity ratio of the reconstituted nucleohistone as a function of UV dose decreased gradually with the increasing histone to DNA weight ratio (r). This effect, was not observed when the histones remained dissociated from DNA, in high ionic strength (1.5 M NaCl). Histone-DNA complex (r=0.97) irradiated up to a dose of 3.6×10⁴ J/m² had a stable melting temperature unlike free DNA where UV irradiation lowered the melting temperature and the heterogeneous melting profiles were observed. Rate of formaldehyde reaction, with DNA recovered from the irradiated complex, was slower than that with native DNA treated at the same dose. All this suggested that the effect of UV in the DNA of the nucleohistone was less, compared to that in free DNA.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10⁴ electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.     

Biochemical and ultrastructural characterizations of nucleoprotamine in human sperm heads treated with micrococcal nuclease and salt

Preparation of human sperm heads containing mainly nucleoprotamines as their chromatin constituent was described. Acrosome-depleted human sperm heads were treated with micrococcal nuclease followed with 2 M NaCl. The treatment specifically removed nucleohistone as small chromatin fragments from the human sperm heads. The nuclease-NaCl-treated sperm head pellets primarily contained nuclease-resistant nucleoprotamine as their chromatin. Transmission electron microscopic study showed that there were two populations of the nuclease-NaCl-treated human sperm head pellets. One population had thick nucleoprotamine cords of ?650–1,200 Å in diameter, and the other had thin nucleoprotamine cords of ?330–420 Å.     

The structure of fibrin. An electron microscopic investigation

An electron microscopic investigation on the structure of fibrin is reported. Fibrin morphology was investigated in a wide variety of experimental conditions, and by carefully controlled staining procedures. Two main band patterns A (230-Å-spaced main dark bands) and B (230-Å-spaced main light bands) are observed for stained fibrin; it is shown that the former results from the superimposition of both positive and negative staining, and the latter is given by positive staining. By suitable denaturation experiments, it was found that the fiber is composed of a regular alternation of lose and dense regions along the axis. We have assumed that the monomer of fibrin is described by the three-nodular model of Hall and Slayter, as supported by recent investigations. The monomers are arrayed according to a head-to-tail sequence along the fiber, and to a staggered lateral association. This model accounts for all the experimental observations, and predicts well the high-resolution band pattern of fibrin. It further agrees with the results of a recent work on the early stages of the fibrinogen-to-fibrin conversion.     

Atomic force microscopy and cytochemistry of chromatin from marsupial spermatozoa with special reference to Sminthopsis crassicaudata

Atomic force microscopy (AFM) of the nuclear topology of spermatozoa from two marsupial species, Sminthopsis crassicaudata and Trichosurus vulpecula was investigated to determine the structural organisation of the chromatin subunits. That of the former species is of special interest as it has a peripheral nucleohistone region (C2) as well as a nuclease-resistant, nucleoprotamine core region (C1). Atomic force microscopy showed that the C2 region contained clusters of 120–160 nm nodules, whereas the C1 region exhibited smaller 50–80 nm nodules. The spermatozoa nuclei of Trichosurus, which has mainly nucleoprotamines, contained higher order chromatin structures of similar size to those from the C1 region of Sminthopsis. This study shows that nucleohistones and nucleoprotamines of marsupial sperm form distinct higher order conformations. For the second part of this work, the chromatin density and affinity for cationic stains of Sminthopsis spermatozoa were determined. Spermatozoa were observed with the transmission electron microscopy (TEM) either unstained or stained with metal salts. In the unstained specimens, the C2 nucleohistone region appeared more electron-lucent than the C1 region. When large cations such as uranyl were used, the reverse situation was observed. Therefore, the electron-dense appearance of the C2 chromatin in conventionally stained material may be due to the presence of net negative DNA charges that attract the cations used for EM staining, whereas the C1 chromatin may lack excess DNA negative charges that attract these stains and thus appears less electron-dense. Mol. Reprod. Dev. 48:367–374, 1997. © 1997 Wiley-Liss, Inc.     

Determination of chemical identity and occupancy from experimental density maps

Three basic electronic properties of molecules, electron density (ED), charge density (CD), and electrostatic potentials (ESP), are dependent on both atomic mobility and occupancy of components in the molecules. Small protein subunits may bind large macromolecular complexes with a reduced occupancy or an increased atomic mobility or both due to affinity‐based functional regulation, and so may substrates, products, cofactors, ions or solvent molecule to the active sites of enzymes. A quantitative theory is presented in this study that describes the dependence of atomic functions on atomic B‐factor in Fourier transforms of the corresponding maps. An application of this theory is described to an experimental ED map at 1.73‐Å resolution, and to an experimental CD map at 2.2‐Å resolution. All the three density functions are linearly proportional to occupancy when the structure factor F(000) term of Fourier transforms of experimental density maps is included. Upon application of this theory to both experimental CD and ESP maps recently reported for photosystem II‐light harvesting complex II supercomplex at 3.2‐Å resolution, the occupancy of two extrinsic protein subunits PsbQ and PsbP is determined to be 20.4 ± 0.2%, and the negative mean ESP value of vitreous ice displaced by the supercomplex on electron scattering path is estimated to be 3% of the mean ESP value of protein α‐helices.     

On the structure of liver ribosomes

The morphology of liver ribosomes and their subparticles, large and small, has been investigated. Analysis of the images has been carried out by successive selection of models and by X-raying them under conditions simulating negative staining. The relation between the main views has been checked by tilting the specimens in an electron microscope through ± 30 °.The small subparticle consists of an elongated body, to one of the ends of which a short “head” is attached. A model has been proposed, whose projections on rotation with respect to the longitudinal axis would satisfy all observable types of images. According to the proposed model, the “head” is tilted with respect to the elongated portion. The length of the subparticle is 230 Å. The dimensions of the elongated portion in the transverse direction are 110 Å × 75 to 80 Å.The large subparticles in lateral view resemble short “rods” 220 to 240 Å long and about 70 to 95 Å wide. At a distance of about 60 Å from the left end of the particles a projection (60 Å in length) is seen, on the inner side of which a depression, or “channel”, filled with the contrasting substance is always observed. Next to this depression a second projection is located, whose height is about 30 Å. The channel is either a cavity in the body of the large subparticle or a part of the RNA without protein. The length of the channel is about 80 Å, the width is about 50 to 60 Å. The left end of the particles is characterized by two sharpened portions; as a result, a cavity that shows an obtuse angle profile makes its appearance. The opposite end of the particles is cut off at an angle of 45 °. In another view, the subparticles appear to be almost rectangular in shape; they are characterized by dimensions of 150 Å × 220 to 240 Å. It is likely that the large projection is displaced sideways with respect to the longitudinal axis of the particles. The asymmetry associated with this displacement gives rise to preferred arrangements of the subparticles on the supporting film. An analysis has been made of the most typical images of monomeric ribosomes, on the basis of which a suggestion is made about mutual orientation of subparticles in a monomer.     

Tuning the redox potential of the primary electron donor in bacterial reaction centers by manganese binding and light-induced structural changes

The influence of transition metal binding on the charge storage ability of native bacterial reaction centers (BRCs) was investigated. Binding of manganous ions uniquely prevented the light-induced conformational changes that would yield to long lifetimes of the charge separated state and the drop of the redox potential of the primary electron donor (P). The lifetimes of the stable charge pair in the terminal conformations were shortened by 50-fold and 7-fold upon manganous and cupric ion binding, respectively. Nickel and zinc binding had only marginal effects. Binding of manganese not only prevented the drop of the potential of P/P⁺ but also elevated it by up to 117 mV depending on where the metal was binding. With variable conditions, facilitating either manganese binding or light-induced structural changes a controlled tuning of the potential of P/P⁺ in multiple steps was demonstrated in a range of ~200 mV without the need of a mutation or synthesis. Under the selected conditions, manganese binding was achieved without its photochemical oxidation thus, the energized but still native BRCs can be utilized in photochemistry that is not reachable with regular BRCs. A 42 Å long hydrophobic tunnel was identified that became obstructed upon manganese binding and its likely role is to deliver protons from the hydrophobic core to the surface during conformational changes.     

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO₂ stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.