Arginine-rich histone

There is a strong electrostatic contribution to the interaction of histones with DNA. Increasing concentration of salt induces the stepwise dissociation of histones from chromatin and nucleosomes. There are three stages in this process. In the first stage, up to 0.7 M NaCl, HI is selectively dissociated. Between 0.7 and 1.2 M NaCl, H2A and H2B are removed, whereas the arginine-rich histones H3 and H4 are dissociated only above 1.2 M NaCl. At 2.0 M NaCl the DNA of chromatin is free of histones (Ohlenbusch et al., 1967 Burton et al., 1975). 

Histones were initially assumed to form part of an assembly system (Sperling and Bustin, 1974, 1975) for two reasons. First, they are present in chromatin as many copies of a few components. [The mass of the five histones equals that of the DNA (for references, see Phillips, 1971 Hnilica, 1972).] Second, fiber X-ray diffraction studies of chromatin (Luzzati and Nicolaieff, 1959 Wilkins et al., 1959 Pardon et al., 1967 Pardon and Wilkins, 1972) indicated that they are involved in a regular periodic structure. The fact that the histones are highly conserved with respect to their primary structure (Dayhoff, 1972 Wilson et al., 1977), especially the arginine-rich histones H3 and H4, also indicates that they may play a primarily structural role in which each residue is crucial. 

All of the core histones share a conserved 65-residue histone fold.27 28 The arginine-rich histones have a strongly conserved amino acid sequence, histone H4 from pea seedlings differing from that of the bovine thymus by only two amino acids. On the other hand, the lysine-rich HI is almost species-specific in its sequence. Differentiated tissues contain at least seven variant forms of histone HI including proteins designated HI0, Hit, and H5 29-31... 

Protein (lysine) Methyltransferase. The enzyme which methylates lysine residues of proteins was named protein methylase III by Paik and Kim (210). Its recommended trivial name is protein (lysine) methyltransferase (S-adenosyl-L-methionine protein-lysine methyltransferase EC 2.1.1.25). Protein (lysine) methyltransferase was found in all rat organs examined and was localized exclusively in the nuclei. Paik and Kim (210) solubilized the enzyme from an acetone powder of calf thymus and purified it 1.3-fold. The enzyme was difficult to work with in the solubilized state, since its activity was lost on overnight storage at either — 10° or 3°C. The enzyme was most effective in methylating histones, especially arginine-rich histone. Denaturation of histone by heating at 100°C for 30 min had no effect on the rate at which protein-lysine) methyltransferase methylated it. Poly lysine and protamine were methylated at slower rates, but horse heart cytochrome c did not serve as substrate. Km for S-adenosyl-L-methionine was 3.0 X 10"6M. 

Histones are susceptible to enzymatic cleavage sol in an HgjSC -HjSOj medium. Infrared spectra de Loze, Compt. Rend. 246, 417 (1958). Lilly, i V in rats of lysine-rich, slightly lysine-rich, and arginine-rich histones 90. 60-70, 60 mg/kg, Starbuck et al, Arch. Int. Pharmacodyn. Ther. 165, 374 (1967). 

The amino acid sequence of the T4 histone (glycine, arginine-rich histone) was confirmed in the laboratory of Busch [67] using somewhat different methods of hydrolysis (only one amino acid, arginine in position 44, was found in a different position, namely 40). Of considerable significance with respect to the position of histones in evolution is the fact that the amino acid sequence of the pea seed histone is identical to that of calf thymus histone. 

Clearly, the ionic bonding between DNA acidic-DNA phosphate group and the basic lysine residues of the histones constitutes a major mode of interaction between the two macromolecules, but ionic bonds are not the only type of bonds between DNA and histones. The finding that urea helps to dissociate the DNA-histone complex, especially in the case of the arginine-rich histones, suggests that hydrophobic bonding may be involved as well [74]. 

Ogawa, Y., Quagliarotti, G., Jordan, J., Taylor, C.W., Starbuck, W.C., Busch, H. Structural analysis of the glycine-rich arginine-rich histone. III. Sequence of the amino-terminal half of the molecule containing the modified lysine residues and the total sequence. J. biol. Chem. 244, 4387-4392 (1969)... 

It has long been known that the amino-acid sequences of the histones H3 and H4 are highly conserved within all organisms, thus implying a critical function. It is now clear that these L-arginine-rich histones are sufficient and necessary for assembly of DNA into a nucleosome-like structure." Two proteins that facilitate this assembly have been found in eukaryotic nuclei (for a review of chromatin assembly see ref. 117). 

Phosphorylation of histones is another mechanism for the neutralization of basic proteins. The phosphorylation reaction occurs after the completion of the histone synthesis. It is reversible. Phosphorylation of the lysine- and arginine-rich histones precedes an increase in RNA synthesis. This was found in transforming lymphocytes, in the regenerating liver and pancreas, etc. Histones and protamines are phosphorylated at the final stages of spermatogenesis (see review of Allfrey et al, 1964, 1972). Specific kinases which participated in the transport of phosphorus groups from ATP to histones are stimulated by cyclic AMP (Allfrey et al., 1972). 

Some investigations supposed that the mechanism of action of various fractions of histones can differ, i.e., the lysine-rich histone I (fl) inhibits transcription when it is associated with DNA the arginine-rich histone III (f3) inhibits RNA-polymerase activity when the two are associated (Spelsberg et al., 1969). 

Sauter and Marquardt followed RNA and protein synthesis as well as changes in the stock of histones in more detail in the two cells in peonies. They found active RNA and protein synthesis in the vegetative cells. Their cytoplasm produced a strong coloration due to arginine-rich histones which were apparently localized on th ribosomes. In the generative cells practically no RNA and protein synthesis could be detected. The... 

Fig. 88. Curve of elution and separation into three fractions of 200-mg arginine-rich histone f2a and by chromatography...

The results of these experiments are given in Tables 4 and 5. They show clearly that the lysine-rich histone possesses a much stronger repressive action than the arginine-rich histone, and the degree of repression exactly matches the lysine content. 

However, when different histones were added to the isolated nuclei, the effect of lysine-rich and arginine-rich histones in these experiments (Allfrey et al., 1963) was opposite to their effect in... 

The special relationship between the th5nmus nuclei and lysine-rich and arginine-rich histones, in contrast to the results obtained by Bonner and co-workers, is evidently attributable to differences in their effect on nuclear metabolism or to technical details. Barr and Butler (1963) obtained results which agreed practically completely with those obtained in Bonner s laboratory, namely that the lysine-rich histone possessed maximal inhibitory properties on DNA-dependent RNA synthesis when DNA from thymocytes was used. Meanwhile, in analogous systems, opposite results were obtained (Bindley, 1963) maximal inhibition of DNA-dependent RNA synthesis by arginine-rich thymus histone. By the use of a different fractionation method (extraction of histones with different concentrations of HCl) (Ord et al., 1965) a fraction richer in lysine was obtained which also had a weaker inhibitory action on DNA-dependent RNA synthesis. 

Littau and co-workers (1965) studied the chromatin structure of thymus lymphocytes by biochemical and electron-microscopic methods. They found that nucleohistone chromatin masses consist of two types of complexes compact and diffuse. The formation of compact complexes, as they showed, is associated with the lysine-rich histone, which forms cross-linkages between the DNA strands. The diffuse histone, in which synthesis of messenger RNA mainly takes place, has no such cross-linkages. It contains arginine-rich histone which is linked to DNA along its strands. 

The hypothesis that it is not the presence or absence of histones, but their chemical modification (acetylation, methylation, phosphorylation) which determines the repressive or active (de-repressed) state of DNA has often been put forward. Phosphorylation of histones takes place through the action of the enzyme histone phosphokinase after synthesis of the histone protein molecule (with the formation of phosphoserine). Phosphorylation is most perceptible in the lysine-rich fraction. Conversely, acetylation of histones, determined by incorporation of labeled acetate, takes place most actively in arginine-rich histone, and a relationship exists... 

Das, C. C., Kaufmann, B. P., Gay, H. (1) Autoradiographic evidence of synthesis of an arginine-rich histone during spermiogenesis in Drosophila melanogaster. Nature (Lond.) 204, 1008—1009 (1964). 

Poly-L-lysine (mol. wt. 25,000) Poly-L-lysine (mol. wt. 2,500) Arginine-rich histone Avidin... 

The DNA is bound to the histone by electrostatic forces, which are large at physiological ionic strength. Assuming that the DNA forms a supercoil with the dimensions of the nucleosome by continuous deformation of the DNA double helix, it can be calculated that 20-28 kcal/ mol of nucleosome are required for formation of the supercoil (Finch et al., 1977). Each octomer of core histones induces super-coiling in closed circular DNA (Fuller, 1971 Crick, 1976). There is conversion between relaxed and nucleosomal DNA. The arginine-rich histones H3 and H4 are necessary and sufficient for the formation of nucleosomes (Camer-ini-Otero et al., 1976 Sollner-Webb et al., 1976) and induce supercoil-ing in closed circular DNA (Camerini-Otero and Felsenfeld, 1977 Bina-Stein and Simpson, 1977). 

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