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The self-association of oligonucleosomal chromatin particles in solution has been studied by light scattering and sedimentation. In the absence of magnesium ions no association is observed. In the presence of 70mM sodium or 2mM magnesium ions mono, di, tri and tetranucleosomes self-associate only if they contain bound histone 1. This association leads to the formation of compact aggregates and is continuous and non-cooperative. The relevance to higher order arrangements of nucleosomes is discussed.

A dipeptide L-lysine-L-phenylalanine is shown to inhibit both cell sickling and the gelation of solutions of sickle-cell haemoglobin. The effect on deoxyhaemoglobin solutions and gels was followed by centrifugation; a progressive inhibition of gelation was observed up to 30 mM Lys-Phe. The haemoglobin concentration at the plateau (26 g/dl) suggests that an effect might be seen in vivo if suitable concentrations of Lys-Phe (about 20 mM) can be maintained in the blood stream. Additional studies of lag time before onset of gelation support this. Oxygen dissociation curves of sickle cells showed an effect of Lys-Phe only after incubation for 3 h before measurement, the P50 decreasing from 51 mmHg (6.8 MPa) to 41 mmHg (5.5 MPa) for cells depleted of 2,3-bisphosphoglycerate. The effect of Lys-Phe on intact sickle cells was more rapid. A marked increase in the number of unsickled cells in the presence of Lys-Phe was observed after only 15 min incubation. This result, together with measurements of uptake both into the cell and onto the cell membrane shows that the compound produces a membrane-mediated antisickling effect in addition to the effect on haemoglobin in solution within the cell. The membrane effect is not due to a change in cell volume. The properties of this dipeptide may be of value in the treatment of impending and early sickle crisis.

Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model.

The secondary structure of encephalomyocarditis (EMC) virus RNA has been studied in situ and in free solution by absorbance-temperature relationships and by circular dichroism (CD). Extracted EMC virus RNA melts reversibly and has a hypochromicity of about 20%; analysis of CD spectra and formaldehyde treatment suggests that approx. 60% of the nucleotides are involved in base-pairing at 25 degrees C. It is shown that the RNA within the virus particle is less structured than when it exists in free solution, being partially stabilized by capsid protein against melting until the virion is disrupted to release the intact RNA. Upon clarification to remove denatured capsid protein, the released RNA gives a melting profile identical with that of phenol-extracted virus RNA. The results suggest that the intact structure of the virus is dependent upon intimate non-covalent bonds between RNA and protein together with hydrophobic bonds between the protein subunits.

Conditions have been found which allow the separation of protein and RNA from Escherichia coli 70-S ribosomes while inhibiting hydrogen exchange in both. Thus the location of masked exchangeable hydrogens can be determined. Tritiated ribosomes were allowed to exchange for a period, at the end of which 25% of the tritium atoms per ribosomes were retained. It was found that at least 80% of them were in the protein. This value corresponds to 60% of the total exchangeable protein hydrogens. Since dissociation of the subunits is not accompanied by detectable conformational changes in the proteins, it is concluded that a large proportion of the ribosomal protein, but little of the RNA, is inaccessible to solvent and therefore buried in the interior of the particle. The presence of slowly-exchanging hydrogens in the ribosomal protein was confirmed by observations of deuterium exchange by infrared spectroscopy. Solvent perturbation of the ultraviolet absorption band of ribosomes was observed, using deuterium oxide as perturbant. A Perturbation-difference spectrum indicating accessible RNA on the 70-S particle was obtained. On dissociation of the subunits the perturbation increases. Separated 50-S and 30-S subunits exhibited difference spectra, which were almost unaffected by magnesium concentration over wide limits, suggesting that the dissociation is accompanied, irrespective of magnesium concentration, by some general loosening of the structure, as also infereed from earlier work on tritium exchange of subunits. The available evidence concerning the distribution of RNA and proteins between the surface and the interior of the ribsome is briefly discussed.

A spectroscopic characterisation is presented of the icosahedral RNA bacteriophage, μ2, and of its separated components. The circular dichroism of the virus is close to the sum of the native RNA and protein components, as defined by the capsid. Thermal absorbance profiles of μ2 and its RNA show that there is no energetically important interaction between double-helical parts of the RNA and the protein. It is suggested that the RNA exists essentially in the native solvated state within the protein shell. Infrared spectra in 2H2O solution are reported. An analysis of the RNA spectrum shows that there is a high degree of base-pairing, estimated at 63±5 % in all, A-U and G-C pairs being comparably represented.

We have described (Richards et al. 1977) a model for the sub-structure of the core particle (140 base pairs) of the nucleosome repeat in chromatin, based principally on results from neutron and X-ray scattering, together with data from other physico-chemical studies. Like that proposed by others (Weintraub, Worcel & Alberts 1976), a principal feature of the model is that it consists of two identical halves, each containing a heterotypic histone tetramer, which lie face-to-face about a dyad axis of symmetry. In this brief contribution we give more detail to proposals already mentioned (Richards et al. 1976, 1977) for the manner in which such a particle might linearize to accommodate the action of either the polymerase or replicase enzymes in transcription and replication, respectively

Publisher Summary This chapter elaborates the substructure of nucleosome core particles. A typical neutron scattering profile from core particles is shown. Such a profile can be analyzed into two regions which includes the central Gaussian region from which radii of gyration may be calculated by application of the Guinier law, the remainder of the profile containing secondary maxima generated by interference effects arising from the shape, internal scattering density fluctuations of the particle, the latter reflecting variation in concentration, and atomic composition within the particle. Varying the H2O/D2O content of the surrounding medium enables scattering profiles to be recorded over a range of contrast conditions for the components of the core particle. Manipulation of contrast may result in the scatter being due predominantly to DNA, or protein. The radii of gyration can be determined both for the DNA, and for the protein domains within the structure.

The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A.

Ribosomal RNA of E. coli contains some 16 4-thiouridylic acid residues per ribosome. These are not available for reaction in the 70S particle until it is dissociated into subunits. All the RNA and protein thiol groups then react with N-ethylmaleimide. Circular dichroism and melting profiles indicate that the 4-thiouridylic acid residues are in base-paired regions of the chain.