Subunit of chromatin - the nucleosome

Basic structure of a nucleosome
On the basis of X-ray diffraction, electron microscopy, nuclease digestion, and chemical cross linking, a model for nucleosome structure was proposed. Properties of this nucleosome model are presented in Table 27.1 and Figure 27.3. The shape of nucleosome, arrangement of histones including HI were also studied. The diameter of nucleosome is 11 nm and its height is 6 nm. The length of DNA around it is 70 nm (~ 67 nm DNA = approx. 200 base pairs, because 34 Å = 3.4 nm = 10 base pairs), which is equivalent to 200 base pairs. The length of DNA varies not only in different, organisms (from 154 bp in a fungus to as high as 260 bp in a sea urchin sperm), but also in different tissues or even in different regions within the same cell of an organism. The length of DNA, however, suggested that DNA should be coiled around nucleosome core particle. Enzyme DNAase I also led to cleavage of DNA suggesting that DNA lies on the surface of nucleosome. It was, therefore, suggested that histones do not cover DNA, but DNA is wrapped around histone core particle. In other words, it was suggested that it will be more appropriate to describe chromatin as string on beads rather than as beads on string. Neutron scattering technique was also used to find out distances of histones and DNA from centre of nucleosome. It was shown that DNA was farther away than proteins from the centre, again suggesting that DNA is not covered by histones, but is wrapped around histone proteins. A simple model of chromatin showing DNA wrapped around histone core is shown in Figure 27.4.



Structural periodicity and super-coiling of DNA in a nucleosome
The structural periodicity of DNA coiled around the histone octamer, was examined by treatment with enzymes DNAase I and DNAase II. Both of these enzymes make single-strand nicks in DNA, so that double stranded DNA structure is not disturbed. But on denaturation following nicking, single stranded DNA fragments are released. It was shown that these cleavage sites are - 10 bases apart and are designated as SI to S13 (SI is ~ 10 bases away from 5' end, S2 is ~ 20 bases away from 5' end, and so on; see Fig. 27.5). Not all sites are cut with equal frequency, so that on electrophoresis a ladder with segments of lengths, which were multiple of 10 were obtained. It was also shown that pairs of sites from SI to S4 or S10 to S13 lie 10 bp apart, while those from S4 to S10 lie 10.7 bp apart.

The above results differ from cleavage of DNA immobilized on a flat surface, where the cutting periodicity reflects structural periodicity (10.5 bp per turn). A variation in cutting periodicity in core DNA of nucleosome (10.0 bp at ends; 10.7 bp in the middle) means that there is variation in structural periodicity of core DNA; in the middle, this is close to the value of DNA in solution, but at the ends it is less tightly screwed.

Much work on the nucleosome has also been conducted in virus SV40 having a circular molecule of DNA, 5200 bp long (1500 nm long). The DNA is packed into a series of nucleosomes, collectively called a mini-chromosome, which is 210 nm long showing a packing ratio of about 7. This minichromosome has been used for a study of supercoiling of DNA in nucleosomes. The DNA in chromatin undergoes supercoiling due to the following reasons : (i) DNA is wrapped around the nucleosome octamer. (ii) Nucleosomes are also coiled using 5-6 nucleosomes per turn. Some of this supercoiling is restrained by the associated proteins (histone octamer). Theoretically, the path of DNA around one nucleosome should generate -1.8 supercoils, if no strain is exercised by proteins. Using SV40 DNA, measurements of supercoiling in DNA involved in nucleosome structure were made which gave a value of -1 turn per nucleosome as against -1.8 expected in the absence of proteins. This discrepancy is sometimes described as linking number paradox (linking number = number of times the two DNA strands are intertwined in circular DNA). The periodicity of 10 bp in DNA wrapped on nucleosome is actually lower than 10.5 bp found in DNA in solution. This allows for absorption of a part of negative supercoils reducing it from -1.8 to -1.0 in the nucleosomes.

Core particle
When nuclease treatment was prolonged beyond the cleavage between nucleosomes, the DNA was removed from both the free ends yielding particles containing DNA of only 146 (or sometimes 140) nucleotide pairs instead of 200 nucleotide pairs (Fig. 27.6). This reduced form of nucleosome is called core particle. Due to nuclease treatment, the length of the DNA is reduced in discrete steps as shown in Figure 27.7. Adjacent core particles, each with core DNA, are joined with each other through DNA segment called linker DNA, which is removed during prolonged digestion. The core DNA has an invariant length of 146 bp, but the linker DNA can vary from 8 bp to 114 bp, thus accounting for variation in DNA length of nucleosomes (154-260 bp).

The core particles could be obtained in crystalline form for a study of X-ray crystallography to reveal the three dimensional structure. Electron microscopy and X-ray diffraction patterns also revealed that nucleosome core particle is neither spherical, nor perfectly flat, but is wedge shaped, about 1] nm in diameter and 6 nm high. The study of crystals suggested that DNA double helix is coiled into a large helix or superhelix, by making two turns around the histones in the middle of the particle. The two turns of the DNA double helix will almost touch, since DNA is 2 nm thick and the particle is only 6 nm thick. From the structure of DNA and the diameter of core particle, it has been estimated that two superhelical turns would have about 160 base pairs (80 bp per turn). Since length of DNA in a core particle is only 146 pairs, it will occupy only one and three-quarter turns. The particle, therefore, will be thinner on one side, accounting for the wedge shape. It was also shown that histone octamer itself is also bipartite and wedge-shaped providing for a short helical ramp on which the DNA is wound (Fig. 27.8).

The closeness of two superhelical turns of DNA on the histone octamer also has a functional consequence. This allows the binding of a protein molecule simultaneously to two different sites of DNA, 80 bp apart. In Expression of Gene : Protein Synthesis 2.  Transcription in Prokaryotes and Eukaryotes and Regulation of Gene Expression 3. A Variety of Mechanisms in Eukaryotes, it will be seen that a promoter site (CAAT box) may lie 80 bp upstream from start point. In such a situation superhelical turns in the nucleosome may allow theassociation of RNA polymerase enzyme to the) promoter and initiation sites simultaneously due to closeness of two turns on the nucleosome octamer. In 1993, in a study of interaction of nucleosome with Xenopus vitellogenin Bl promoter,, it was shown that the presence of nucleosome stimulates transcription by oestrogen. It is believed, that by DNA looping, the oestrogen receptor binding site is brought closer to the site of transcription complex formation.