I.O. Walker

Studies on the DNA of a virus from Galleria mellonella

A small spherical virus, Galleria Free virus, (GFV), and empty protein shells (top component) have been isolated from the insect, Galleria mellonella. GFV has been shown to contain DNA [l] . We wish to describe some unusual features of the DNA which appears to have a partially single-stranded conformation in the virus and which, when isolated, adopts a highly ordered structure characteristic of native double-stranded DNA.

Covalent cross‐linking of histones in chromatin

There is currently much interest in the possibility that chromatin (DNH) may consist of a repeating nucleoprotein subunit. The idea is supported by the in vivo degradation of DNA in chromatin to a series of discrete size classes [l] and by recent studies on isolated histones which report specific interactions between fractions [2-41. Furthermore, studies in this laboratory have suggested that in the intact chromatin the histones are arranged in a regular, repeating manner along the DNA [5-81. To obtain more direct information about the spatial relationships between the histones in chromatin, we have c>valently crosslinked the proteins using formaldehyde [9]. To aid the interpretation of the results we have cross-linked not only native DNH but also DNH which is specifically depleted of fl and which is still in the supercoil conformation [ 10,l l] or of fl, f2a2 and f2b where the rigid supercoil properties have been lost [ 121. The nucleoproteins have also been treated with formaldehyde in different NaCl concentrations to study the effects of conformational changes and differential dissociation of histones on the reaction. The products have been examined by polyacrylamide-SDS gel electrophoresis. The results show that when dissociated from DNA in high salt f2al and f3 undergo extensive polymerisation, the polymerising unit being a dimer of f2al and f3. When bound to DNA f2al and f3 can interact extensively to form high mol. wt polymers (apparent mol. wt 170 000 and above) with the dimer again being the basic polymerising unit. Similar behaviour is found for f2a2 and f2b although the two polymerising systems do not appear to interact with each

Structural studies on ribosomes II. Denaturation and sedimentation of ribosomal subunits unfolded in EDTA

Abstract The effect of removing Mg2+ from 30- and 50-S ribosomal subunits and ribosomal RNA by the direct addition of EDTA at room temperature has been followed using the analytical ultracentrifuge. 50-S subunits unfolded by a cooperative process, without the dissociation of the protein and RNA moieties, via two discrete, partially unfolded species, to give a 16-S particle. Ribosome-like particles were reformed when the unfolded species were dialysed into magnesium acetate. The unfolding process could be represented as follows: 50 S ⇌ 35 S ⇌ 21 S ⇌ 16 S The unfolded ribosomes showed a limited polyelectrolyte behaviour on altering the Na+ concentration or removing Mg2+ and a tendency to specifically refold as the Na+ concentration was increased. By contrast the sedimentation coefficient of 23-S RNA showed progressive changes as Mg2+ was removed with EDTA. No discrete intermediate species were formed. The native 50-S ribosome and the unfolded species had the same total amount of secondary structure and the denaturation behaviour of the 16-S species was the same as 23-S RNA in EDTA. In the unfolded state therefore, the protein did not affect the denaturation behaviour of the RNA. The RNA in the native ribosome was, however, more stable to denaturation than 23-S RNA. This stabilisation was attributed to the tertiary structure of the ribosome. These and other hydrodynamic considerations implied that protein interactions played an important part in maintaining the tertiary structure of the 50-S subunit and suggested that protein was associated with the interhelical regions of the RNA. More limited studies on the 30-S subunits showed that these unfolded to give an 18-S particle without dissociation of the protein and RNA. Under certain conditions the secondary structure of the 18-S species was less than that in the intact ribosome. The thermal denaturation behaviour of the native 30-S ribosome when compared with 16-S RNA showed that, as with the 50-S ribosome, the tertiary structure was stabilised by protein interactions, the protein being associated with the interhelical regions on the RNA.

The effect of proflavine on helix-coil transitions in sodium deoxyribonucleate and deoxyribonucleohistone.

Abstract The spectrum of proflavine has been examined in the presence of DNA, deoxyribonucleohistone, apurinic acid and sodium poly- l -glutamate. A weak, electrostatic binding process exemplified by the interaction of proflavine with sodium poly- l -glutamate results in a uniform decrease in absorption in the visible range and a slight hypochromism in the ultraviolet region. A strong binding process, involving both electrostatic and Van der Waals forces occurs when proflavine interacts with DNA, heat- or acid-denatured DNA, nucleohistone or apurinic acid. This is characterised by a displacement of the spectrum in the visible range to longer wavelengths and a decrease in absorption in the ultraviolet region. The hypochromism is explained in terms of an interaction between the planar dye molecules and the planar bases on the DNA or apurinic acid and in the case of nucleohistone, between the dye and the bases on the DNA moiety. The binding of proflavine to either DNA or DNH stabilises the macromolecule towards thermal denaturation. The magnitude of the effect depends on the amount of dye bound and reaches an upper limit at low DNA/dye mole ratios which appears to correspond to the upper limit of strong binding. Stabilisation is not observed in solutions of high ionic strength. Theoretical considerations show that the stabilisation is consistent with the intercalation of proflavine molecules between the nucleotide bases of the DNA or deoxyribonucleohistone.

Structural studies on ribosomes. I. The binding of proflavine to Escherichia coli ribosomes.

Abstract The binding of proflavine to 30-S, 50-S and 70-S ribosomes has been studied quantitatively utilising spectral changes in the dye to measure the degree of binding. All three particles produced hypochromism in the ultraviolet difference spectrum of the dye and a displacement in the visible spectrum to longer wavelengths with the formation of characteristic isobestic points. An analysis of the spectral changes revealed two distinct binding processes. A strong binding characterised by a free energy of association of 10 kcal/mole appeared to involve Van der Waals interactions between the dye and the bases on the RNA; a weak interaction was attributed to electrostatic binding of the dye to the RNA phosphates. The number of strong binding sites was 96, 70 and 108 for the 30-S, 50-S and 70-S ribosomes, respectively. Despite an increase of about 50 in the number of binding sites when 70-S ribosomes dissociate into their subunits the binding of proflavine to 30-S and 50-S ribosomes did not appear to affect the reassociation of these particles to form 70-S ribosomes. The number of strong binding sites on the 50-S ribosome increased to about 500 when the particle was unfolded in EDTA. This suggested that binding sites, which are exposed in the unfolded particle, are not available for binding dye in the intact ribosome because of the rigid tertiary folding in the native particle.

The effect of ionic strength of helix-coil transitions in nucleic acid

Abstract The thermal denaturation of the nucleic acid is considered as a first-order phase change, the transition temperature of which is determined only by the ionic strength of the solvent and the free energy. An equation is derived which relates the transition temperature to ΔG E , the differene in electrostatic free energy between the native and denatured nucleic acids. The adoption of cylindrical models for both native and denatured nucleic acid enables ΔG E to be estimated at various ionic strengths. Application of the theory then yields estimates for the enthalpy and entropy of denaturation and the free energy of the stabilising forces in the helix.

Control of RNA transcription in nuclei and nucleoli of Physarum polycephalum.

RNA synthesis in nuclei isolated from synchronised cultures of Physarum polycephalum shows an apparent biphasic pattern over the mitotic cycle which implies that there may be control over the transcriptional activity of both polymerases A and B [ 1,2] . Using a modified method of isolating both nuclei and nucleoli, we have recently demonstrated that the size-distribution of RNA synthesised in vitro using the endogenous polymerase activities is very similar to that synthesised in vivo. In nucleoli it appears that rRNA (ribosomal RNA) precursors are correctly re-initiated, synthesised and processed [3]. We thought it worthwhile, therefore, to re-investigate the specific activity of the endogenous polymerases present in both nuclei and nucleoli during the mitotic cycle using a-amanitin to inhibit the nucleoplasmic polymerase B and the rifamycin derivative AF/013 to inhibit reinitiation [4]. The results show that whereas polymerase B activity shows a maximum in mid-S phase, decreasing to a constant value for the rest of interphase, the nucleolar polymerase A activity remains constant throughout interphase even though the number of genes (template) and the number of polymerase molecules doubles during the mitotic cycle. Two possible control mechanisms are discussed.

Histones F2a1 and F3 interact reversibly and cooperatively with DNA to form an equimolar complex in chromatin

Recent studies on isolated histones in solution have highlighted the importance of specific interactions between f2al and f3 to give dimers, tetramers and higher oligomers [1-4] . Such studies take on a new significance with the demonstration that similar, though more extensive interactions also occur in chromatin where the histones are bound to DNA [4]. A model incorporating these observations has recently been proposed for chromatin structure [5]. Here we present data which demonstrate that f2al and t3 dissociate reversibly and cooperatively as an equimolar complex from calf thymus chromatin. This study complements previous results which showed that in native chromatin f2al and f3 can be covalently cross-linked into oligomeric products containing equimolar quantities of the two histones [4]. The cooperative reversible interaction of f2al and f3 with DNA provides a mechanism whereby the histones can be self-assembled into the highly ordered oligomers which may be an important feature of the structure of the chromatin supercoil [5].

Structural studies on Escherichia coli ribosomes: III. Denaturation and sedimentation of ribosomal subunits unfolded in urea

Abstract The addition of urea to 50-S ribosomal subunits caused a lowering of the sedimentation coefficient to 32–38 S by a one-step co-operative process. This was interpreted as an unfolding of the native ribosome caused by the disruption of hydrophobic bonds between ribosomal proteins. In many cases slow-moving components were evident in the ultracentrifuge which were probably dissociated protein. Treatment of 50-S ribosomes with formaldehyde prevented the dissociation of the protein from RNA on subsequent reaction with urea. The ribosomes appeared to unfold to give slower-sedimenting species but the nature of the unfolding was different from that of untreated ribosomes. Thus the ribosomes were more sensitive to reaction with urea and the unfolding was no longer highly co-operative. The addition of varying amounts of EDTA to formaldehyde-treated ribosomes produced a single unfolded component with a sedimentation coefficient of 34–38 S. Unlike untreated ribosomes no further unfolding occurred, either with time or with a large excess of EDTA. No loss of secondary structure in the RNA occurred when native or formaldehyde-treated ribosomes were reacted with urea or EDTA at room temperature. The melting temperatures decreased in urea and EDTA and the melting curves became less co-operative. The melting profiles of unfolded ribosomes in urea and EDTA were similar to RNA in the same solvent and showed that in the unfolded form the protein had little effect on the thermal denaturation properties of the RNA. It is concluded that in the unfolded ribosomes, characterised by a sedimentation coefficient of 30–35 S, the major bonds involved in the maintenance of ribosomal tertiary structure have been broken. These bonds involve interactions between the proteins of the ribosome.

Studies on the properties of tryptophan and tyrosine side chains in tobacco mosaic virus and tobacco mosaic virus protein

Two of the three tryptophan residues in each tobacco mosaic virus (TMV) protein subunit were oxidised with N-bromosuccinimide at pH 7.0. Oxidation of these residues did not impair the ability of the subunits to polymerise into protein rods. On the other hand, in the virus or native protein rods the tryptophan residues were not oxidised by N-bromosuccinimide. Thus, in the protein subunit, one tryptophan residue is ‘buried’ and two are situated on the surface but in the virus or protein rod, these latter two residues are situated in regions of interaction between subunits. However, they are not necessarily directly involved in bonding interactions. Solvent perturbation studies showed that the equivalent of at least one tryptophan chromophore was exposed on the surface of the subunit. At least three of the four tyrosine residues in each subunit were titrated reversibly in the native protein with an apparent pK of 10.6. The hydroxyl groups of these residues are probably on the surface of the subunit. Solvent perturbation studies showed that the equivalent of at least two tyrosine chromophores were exposed on the surface of the subunit. In the virus the tyrosine residues titrated irreversibly over a higher range of pH with an apparent pK of 11.0. The titration was paralleled by the disaggregation of the virus particle into subunits. It is suggested that tyrosine residues may be directly involved in quaternary interactions between subunits.