Differential dissociation of histone tails from core chromatin.

The dissociation of the trypsin-sensitive basic tails of the core histones in core chromatin has been followed as a function of [NaCl] using proton NMR spectroscopy. The tails dissociate in a highly cooperative all or none manner over the salt concentration range 0.2-0.6 M, that is, below the salt concentration required to dissociate the complete molecule. Assuming that each basic tail dissociates independently, the total number of salt linkages involved in binding the tails to DNA is 103. This equals the number of basic side chains in the tails of an octamer. The standard free energy of dissociation, delta G degree, in 1 M NaCl at 297 K is 3.6 kcal/mol. Temperature had no effect on the extent of dissociation up to 45 degrees C. However, between 45 and 65 degrees C, where the premelting transition in the core chromatin occurs, the tails dissociated completely. Dissociation of the tails was associated with a conformational transition in the DNA consistent with loss of supercoiling. From this, and the results of a previous study, it can be shown that the structured, trypsin-resistant domain of each core histone octamer makes 100 salt linkages to DNA. Thus, in 10 mM salt, each core octamer makes a total of 203 salt linkages to DNA.

The thermal denaturation of chromatin core particles.

The thermal denaturation of chicken erythrocyte core particles has been followed using changes in absorption and circular dichroism. The absorption-denaturation curve is biphasic, with transitions centred at 59 degrees C and 74 degrees C. The first of these transitions is reversible, whereas heating into the second transition produces irreversible changes in DNA and histone secondary structure. After heating to temperatures within the second transition and then cooling, two components are observed in the analytical centrifuge: a slow-sedimenting species which is present in proportions corresponding to the extent of irreversible denaturation and a fast sedimenting species which is present in inverse proportion to the extent of denaturation. The slow-sedimenting component was primarily denatured DNA. The results suggest that thermal denaturation of core particles is a cooperative, 'all-or-none' process.

Trypsin digestion of core chromatin.

Chicken erythrocyte core chromatin was digested with trypsin for 18 h. Five major limit peptides were produced with mol.wts. from 10 800 to 8000 which arose from the central regions of each core histone. The basic amino- and carboxyl-terminal regions of each core histone were digested to small peptides having an average size of less than six amino acids. The small basic peptides, 25% by weight of the total histone, dissociated from the complex and could be removed by dialysis. The five major limit peptides remained bound to the DNA and contained all the secondary structure originally present in the native histones. Trypsin digestion decreased the supercoiling of the DNA in the complex and perturbed the tertiary structure of the histones. By contrast, there were no changes in the secondary structure of the large degraded histone fragments. However, when these were dissociated from the DNA, the secondary structure, which is predominantly alpha-helix, decreased by 50%. It is concluded that DNA binds strongly to the central regions of the core histones via alpha-helical segments on the polypeptide chains.

Control of histone gene expression in Physarum polycephalum. I. Protein synthesis during the cell cycle.

Synchronous cultures of Physarum polycephalum were pulsed with [3H]lysine hydrochloride in S and G2 phases of the cell cycle. Plasmodial extracts were separated into nuclear, ribosomal and acid-soluble post-ribosomal cytoplasmic fractions. Core histones could be detected by staining in the nuclear fractions of both S and G2 phases, but were not detected by staining in the cytoplasmic fractions. Newly synthesized histone was present in S-phase nuclei but not in S-phase cytoplasm. The specific activity of newly synthesized histone in G2-phase nuclei decreased by at least 95% compared to S phase and no newly synthesized histone was observed in G2-phase cytoplasmic fractions. Thus histone synthesis is restricted to S phase. There are no free pools of histone in the cytoplasm of Physarum in either S or G2 phases of the cell cycle.

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.

The characterisation of a 5-S 'monosome' fraction from chromatin of Physarum polycephalum.

A so-called '5-S mononucleosome' fraction was isolated from nuclei of Physarum polycephalum by digestion with micrococcal nuclease and subsequent fractionation by gel filtration. This fraction had electrophoretic and sedimentation properties which were similar to DNA of approximately 140 base pairs in length. It is shown that lysis of the nuclei activates a protease which is resistant to phenyl-methyl sulphonyl fluoride, o-phenanthroline and parachloromercuri benzene sulphonate and which subsequently degrades the nucleosomes.

Densonucleosis virus structural proteins.

The protein coats of two densonucleosis viruses (types 1 and 2) were examined by a variety of biophysical, biochemical, and serological techniques. The viruses were 24 nm in diameter, contained at least four polypeptides, were remarkably stable to extremes of pH and denaturing agents, and were serologically closely related. The two viruses could, however, be distinguished serologically and by differences in migration of their structural polypeptides. For each virus the "top component" (i.e., the protein coat minus DNA, found occurring naturally in infections) appeared to have a composition identical to that of the coat of the virus and was a more stable structure. Electrometric titration curves of the virus particles and top components demonstrated that the DNA phosphate in densonucleosis virus particles was neutralized by cations other than basic amino acid side chains of the protein coat. Circular dichroism studies showed that there was a conformational difference between the protein coats of top components and virus particles.

The binding of histones H1 and H5 to chromatin in chicken erythrocyte nuclei.

The binding curves of histones H1 and H5 to chromatin in nuclei have been determined by a novel method which utilises the differential properties of free and bound histones on cross-linking with formaldehyde. The dissociation is thermodynamically reversible as a function of [NaCl]. The binding curves are independent of temperature over the range 4 degrees - 37 degrees C and independent of pH over the range 5.0 to 9.0. The curves are sigmoid, indicating co-operative dissociation with NaCl. The standard free energy of dissociation in 1 M NaCl for H1 is 0.5 Kcals/mole and for H5 is 3.5 Kcals/mole.

The interaction of core histones with DNA: equilibrium binding studies.

The binding of core histone proteins to DNA, measured as a function of [NaCl[ is a reversible process. Dissociation and reassociation occurs in two stages. Between 0.7 and 1.2 M NaCl H2a H2b bind non-cooperatively as an equimolar complex with deltaGo = 1.6 Kcals/mole at 4 degree C and 1.0 M NaCl. Between 1.2 and 2.0 M NaCl H3 and H4 bind cooperatively as an equimolar complex with delta Go = 7.4 Kcal/mole at 4 degree C and 1.0 M NaCl. The proper binding of H2a and H2b requires the presence of bound H3 and H4. Nuclease digestion of the H3-H4 DNA produces a tetramer of H3-H4 bound to fragments of DNA 145, 125 and 104 base pairs long. Thus an H3-H4 tetramer can protect fragments of DNA as long as those found in complete core particles and must therefore span the nucleosome core particle.

The structure of nucleolar chromatin in Physarum polycephalum.

The nucleolar DNA of Physarum polycephalum has been differentially labelled with 3H-thymidine and the structure of the nucleolar chromatin investigated by digestion with micrococcal nuclease. Nucleolar chromatin which had been labelled in G2 phase of the cell cycle and then digested before mitosis had an identical DNA repeat length to main band DNA (165 +/- 5 base pairs) but there was a definite indication that the rate of digestion was faster for nucleolar DNA than for main band DNA. Nucleolar chromatin which had been labelled in G2 and the label chased through mitosis into G2 phase of the next cycle showed an identical DNA repeat length to main band DNA and was also digested at the same rate. We conclude that nucleolar chromatin, at least 25 per cent of which is maximally transcriptionally active in G2, has a nucleosome-like structure.

Studies on pulse-labelled RNA during the mitotic cycle of Physarum polycephalum by subnuclear fractionation.

A method is described for the isolation of pulse-labelled RNA from nuclei and subnuclear fractions of Physarum polycephalum. At all times during interphase the nucleolar RNA consisted mainly of a 34-S rRNA precursor with only small amounts of 26-S and 19-S rRNAs. The nucleoplasmic RNA consisted of predominantly mature 26-S rRNA with small amounts of 19-S rRNA and a 30-S RNA species. The 30-S RNA component displayed different labelling kinetics from the rRNA precursors. A low molecular weight RNA fraction (4-8 S) appeared to accumulate in the nucleus as interphase progressed.

In vitro transcription of RNA in nuclei, nucleoli and chromatin from physarum polycephalum.

Methods for isolating nuclei, nucleoli and chromatin from Physarum polycephalum which retain high levels of endogenous RNA polymerase activity are described. Under carefully controlled conditions with respect to mono- and divalent cation concentrations RNA synthesis in nuclei displayed linear kinetics for at least 30 min and the RNA products had a similar size distribution to nuclear RNA synthesis observed in vivo. Chromatin showed 60% of the nuclear transcriptional activity but no conditions were found where faithful transcription of the template occurred. Isolated nucleoli were 5-fold more active than nuclei and the endogenous RNA polymerase activity was insensitive to alpha-amanitin. Under carefully controlled conditions, the nucleoli appeared to support the accurate transcription, re-initiation and processing of rRNA chains in vitro.

The application of ESR to chromatin. Spin-labelling of the histone H3 cysteine residues in situ.

The possibility has been investigated of selectively spin-labelling the cysteine residues of histone H3 in chromatin and probing by ESR conformational changes affecting the labelled area as the molecular environment is altered. About 90% of bound labels are attached to the thiol groups and are strongly immobilized in deep crevices. The remaining labels are bound to amino groups mainly on histone H1, giving rise to a more mobile component in the chromatin spectrum. No conformational changes involving the labelled cysteins could be detected as the histones were dissociated stepwise from the complex by NaCl, but treatment with urea led to a cooperative increase in mobility, indicating that the hydrophobic region around the cysteine residues is folded in a compact tertiary structure to which histone H4 may be bound in the native complex, but which is not affected by dissociation of the H3-H4 unit from the DNA. In addition, chymotryptic disruption of the chromatin has been followed and an estimate made from the rotational correlation times of the size and origin of the digestion fragment carrying spin-labelled cysteine 110.

DNA contained by two densonucleosis viruses.

The DNA contained by particles of densonucleosis viruses 1 and 2 were analyzed within the particle, and properties of DNA extracted from these particles were determined. The DNA appears to exist as a single-stranded molecule with limited secondary structure within particles, as assessed by spectral changes induced by formaldehyde, melting profiles, and circular dichroism studies. The single-stranded DNA had an apparent molecular weight of 1.9 X 10(6) to 2.2 X 10(6) as assessed by differences in the molecular weight of virus particles and top component and percentage of nucleic acid. DNA extracted from virus particles in low-salt buffers possessed properties typical of a single-stranded molecule. Double-stranded DNA could be extracted from virus particles under appropriate high salt and elevated temperature. The linear double-stranded DNA extracted from both viruses had a molecular weight of about 3.9 X 10(6) to 4.1 ZX 10(6) determined by neutral sedimentation and electron microscopy and an equivalent genome size determined by reassociation kinetics. About 87% of the DNA was homologous between the two viruses.

The conformation of 16S RNA in the 30S ribosomal subunit from Escherichia coli.

The digestion of E. coli 16S RNA with a single-strand-specific nuclease produced two fractions separable by gel filtration. One fraction was small oligonucleotides, the other, comprising 67.5% of the total RNA, was highly structured double helical fragments of mol. wt. 7,600. There are thus about 44 helical loops of average size corresponding to 12 base pairs in each 16S RNA. 10% of the RNA could be digested from native 30S subunits. Nuclease attack was primarily in the intraloop single-stranded region but two major sites of attack were located in the interloop single-stranded regions. Nuclease digestion of unfolded subunits produced three classes of fragments, two of which, comprising 80% of the total RNA, were identical to fragments from 16S RNA. The third, consisting of 20% RNA, together with an equal weight of peotein, was a resistant core (sedimentation coefficient 7S).

Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions.

Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.

A model for chromatin sub-structure incorporating symmetry considerations of histone oligomers.

Symmetry considerations of the kind of structures which can be generated when dimers of histones f2al-f3 and f2a2-f2b interact lead to the following conclusions: chromatin subunits based on closed-shell structures give rise to discrete, non-interacting nucleoprotein subunits with the histones arranged at random along the DNA chain; open structures based on infinite helices give rise to highly ordered, regular arrangements of dimers. A model is proposed in which helical polymers of f2al-f3 and f2b-f2a2 form a central core with the DNA helically arranged around it. The helical repeat contains 9.6 turns of B-form DNA and one molecule each of f2al, f2a2, f2b, f3 and f1. The pitch of the helix is 53Angstrom and the other diameter 130Angstrom. The protein molecular repeat is 106Angstrom.