Chromatin structure and chemical composition. Structural organization of chromatin

24.05.2020

The genetic material of eukaryotic organisms has a very complex organization. DNA molecules located in the cell nucleus are part of a special multicomponent substance - chromatin.

Concept definition

Chromatin is the material of the cell nucleus containing hereditary information, which is a complex functional complex of DNA with structural proteins and other elements that provide packaging, storage and implementation of the karyotic genome. In a simplified interpretation, this is the substance that makes up chromosomes. The term comes from the Greek "chrome" - color, paint.

The concept was introduced by Fleming back in 1880, but there is still debate about what chromatin is in terms of biochemical composition. Uncertainty concerns a small part of the components that are not involved in the structuring of genetic molecules (some enzymes and ribonucleic acids).

In an electron photograph of the interphase nucleus, chromatin is visualized as numerous areas of dark matter, which can be small and scattered or unite into large dense clusters.

The condensation of chromatin during cell division results in the formation of chromosomes that are visible even under a conventional light microscope.

Structural and functional components of chromatin

In order to determine what chromatin is at the biochemical level, scientists extracted this substance from cells, transferred it into a solution, and in this form studied the component composition and structure. In this case, both chemical and physical methods were used, including electron microscopy technologies. It turned out that chemical composition 40% of chromatin is represented by long DNA molecules and almost 60% by various proteins. The latter are divided into two groups: histones and nonhistones.

Histones are a large family of basic nuclear proteins that bind tightly to DNA, forming the structural skeleton of chromatin. Their number is approximately equal to the percentage of genetic molecules.

The rest (up to 20%) of the protein fraction falls on DNA-binding and spatially modifying proteins, as well as enzymes involved in the processes of reading and copying genetic information.

In addition to the main elements, ribonucleic acids (RNA), glycoproteins, carbohydrates, and lipids are found in small amounts in chromatin, but the question of their association with the DNA packaging complex is still open.

Histones and nucleosomes

The molecular weight of histones varies from 11 to 21 kDa. A large number of residues of the basic amino acids lysine and arginine give these proteins a positive charge, contributing to the formation ionic bonds with oppositely charged phosphate groups on the DNA double helix.

There are 5 types of histones: H2A, H2B, H3, H4 and H1. The first four types are involved in the formation of the main structural unit of chromatin - the nucleosome, which consists of a core (protein core) and DNA wrapped around it.

The nucleosomal core is represented by an octameric complex of eight histone molecules, which includes the H3-H4 tetramer and the H2A-H2B dimer. A stretch of DNA with a length of about 146 nucleotide pairs is wound onto the surface of a protein particle, forming 1.75 turns, and passes into a linker sequence (approximately 60 bp) that connects nucleosomes to each other. The H1 molecule binds to the linker DNA, protecting it from the action of nucleases.

Histones can undergo various modifications such as acetylation, methylation, phosphorylation, ADP-ribosylation, and interaction with the ubivictin protein. These processes affect the spatial configuration and packing density of DNA.

Non-histone proteins

There are several hundred varieties of non-histone proteins with different properties and functions. Their molecular mass varies from 5 to 200 kDa. A special group is made up of site-specific proteins, each of which is complementary to a specific DNA region. This group includes 2 families:

"zinc fingers" - recognize fragments with a length of 5 nucleotide pairs;
homodimers - characterized by the structure "helix-turn-helix" in the fragment associated with DNA.

The best studied are the so-called high mobility proteins (HGM proteins), which are permanently associated with chromatin. The family received this name because of the high speed of movement of protein molecules in an electrophoretic gel. This group occupies the majority of the non-histone fraction and includes four main types of HGM proteins: HGM-1, HGM-14, HGM-17, and HMO-2. They perform structural and regulatory functions.

Non-histone proteins also include enzymes that provide transcription (the process of messenger RNA synthesis), replication (doubling of DNA) and repair (elimination of damage in the genetic molecule).

Levels of DNA compaction

The peculiarity of the structure of chromatin is such that it allows DNA strands with a total length of more than a meter to fit into a nucleus with a diameter of about 10 microns. This is possible due to the multi-stage packaging system of genetic molecules. General scheme compaction includes five levels:

nucleosomal thread with a diameter of 10–11 nm;
fibril 25–30 nm;
loop domains (300 nm);
fiber 700 nm thick;
chromosomes (1200 nm).

This form of organization reduces the length of the original DNA molecule by 10,000 times.

A thread 11 nm in diameter is formed by a number of nucleosomes connected by linker regions of DNA. In an electron micrograph, such a structure resembles beads strung on a fishing line. The nucleosomal filament is coiled like a solenoid, forming a fibril 30 nm thick. Histone H1 is involved in its formation.

The solenoid fibril folds into loops (in other words, domains), which are fixed on the supporting intranuclear matrix. Each domain contains from 30 to 100 thousand base pairs. This level of compaction is characteristic of interphase chromatin.

A structure with a thickness of 700 nm is formed during the spiralization of a domain fibril and is called a chromatid. In turn, two chromatids form the fifth level of DNA organization - a chromosome with a diameter of 1400 nm, which becomes visible at the stage of mitosis or meiosis.

Thus, chromatin and chromosome are forms of packaging of genetic material that depend on the life cycle of the cell.

Chromosomes

The chromosome consists of two sister chromatids identical to each other, each of which is formed by one supercoiled DNA molecule. The halves are connected by a special fibrillar body called the centromere. At the same time, this structure is a constriction dividing each chromatid into arms.

Unlike chromatin, which is a structural material, a chromosome is a discrete functional unit characterized not only by structure and composition, but also by a unique genetic set, as well as a certain role in the implementation of the mechanisms of heredity and variability at the cellular level.

Euchromatin and heterochromatin

Chromatin in the nucleus exists in two forms: less coiled (euchromatin) and more compact (heterochromatin). The first form corresponds to the transcriptionally active regions of DNA and therefore is not so densely structured. Heterochromatin is divided into facultative (it can change from an active form to a dense inactive one, depending on the stage of the cell's life cycle and the need to realize certain genes) and constitutive (constantly compacted). During mitotic or meiotic division, all chromatin is inactive.

Constitutive heterochromatin is found near the centromere and at the ends of the chromosome. The results of electron microscopy show that such chromatin retains a high degree condensation not only at the stage of cell division, but also during interphase.

The biological role of chromatin

The main function of chromatin is to tightly pack large amounts of genetic material. However, it is not enough just to fit DNA in the nucleus for the life of the cell. It is necessary that these molecules "work" properly, that is, they can transmit the information contained in them through the DNA-RNA-protein system. In addition, the cell needs to distribute genetic material during division.

The structure of chromatin fully meets these tasks. The protein part contains all the necessary enzymes, and the structural features allow them to interact with certain sections of DNA. Therefore, the second important function of chromatin is to provide all the processes associated with the implementation of the nuclear genome.

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Structure and chemistry of chromatin

Chromatin is a complex mixture of substances from which eukaryotic chromosomes are built. The main components of chromatin are DNA and chromosomal proteins, which include histones and non-histone proteins, which form structures highly ordered in space. The ratio of DNA and protein in chromatin is ~1:1, and the bulk of chromatin protein is represented by histones. The term "X" was introduced by W. Flemming in 1880 to describe intranuclear structures stained with special dyes.

Chromatin- the main component of the cell nucleus; it is fairly easy to obtain from isolated interphase nuclei and from isolated mitotic chromosomes. To do this, use its property to go into a dissolved state during extraction aqueous solutions with low ionic strength or simply deionized water.

Chromatin fractions obtained from different objects have a fairly uniform set of components. It was found that, in terms of total chemical composition, chromatin from interphase nuclei differs little from chromatin from mitotic chromosomes. The main components of chromatin are DNA and proteins, among which the bulk are histones and non-histone proteins.

Slide3 . There are two types of chromatin: heterochromatin and euchromatin. The first corresponds to the sections of chromosomes condensed during interphase, it is functionally inactive. This chromatin stains well; it is this chromatin that can be seen on the histological preparation. Heterochromatin is divided into structural (these are sections of chromosomes that are constantly condensed) and facultative (it can decondense and turn into euchromatin). Euchromatin corresponds to decondensation in interphase regions of chromosomes. This is a working, functionally active chromatin. It does not stain, it is not visible on the histological preparation. During mitosis, all euchromatin is condensed and incorporated into chromosomes.

On average, about 40% of chromatin is DNA and about 60% is proteins, among which specific nuclear histone proteins make up 40 to 80% of all proteins that make up isolated chromatin. In addition, the composition of chromatin fractions includes membrane components, RNA, carbohydrates, lipids, glycoproteins. The question of how these minor components are included in the structure of chromatin has not yet been resolved. Thus, the RNA may be a transcribed RNA that has not yet lost its association with the DNA template. Other minor components may refer to the substances of the coprecipitated fragments of the nuclear envelope.

PROTEINS are a class of biological polymers present in every living organism. With the participation of proteins, the main processes that ensure the vital activity of the body take place: respiration, digestion, muscle contraction, transmission of nerve impulses.

Proteins are polymers, and amino acids are their monomer units.

Amino acids - it organic compounds containing in their composition (in accordance with the name) an amino group NH2 and an organic acid, i.e. carboxyl, COOH group.

A protein molecule is formed as a result serial connection amino acids, while the carboxyl group of one acid interacts with the amino group of the neighboring molecule, as a result, a peptide bond is formed - CO-NH- and a water molecule is released. Slide 9

Protein molecules contain from 50 to 1500 amino acid residues. The individuality of a protein is determined by the set of amino acids that make up the polymer chain and, no less important, by the order of their alternation along the chain. For example, the insulin molecule consists of 51 amino acid residues.

Chemical composition of histones. Peculiarities physical properties and interaction with DNA

Histones- relatively small proteins with a very large proportion of positively charged amino acids (lysine and arginine); the positive charge helps the histones bind tightly to the DNA (which is highly negatively charged) regardless of its nucleotide sequence. The complex of both classes of proteins with the nuclear DNA of eukaryotic cells is called chromatin. Histones are unique characteristic eukaryotes and are present in huge numbers per cell (about 60 million molecules of each type per cell). Histone types fall into two main groups, nucleosomal histones and H1 histones, forming a family of highly conserved basic proteins, consisting of five large classes - H1 and H2A, H2B, H3 and H4. H1 histones are larger (about 220 amino acids) and have been found to be less conserved over the course of evolution. The size of histone polypeptide chains ranges from 220 (H1) to 102 (H4) amino acid residues. Histone H1 is highly enriched in Lys residues, histones H2A and H2B are characterized by a moderate content of Lys, the polypeptide chains of H3 and H4 histones are rich in Arg. Within each histone class (with the exception of H4), several subtypes of these proteins are distinguished based on amino acid sequences. This multiplicity is especially characteristic of mammalian H1 class histones. In this case, seven subtypes are distinguished, named H1.1-H1.5, H1o and H1t. Histones H3 and H4 are among the most conserved proteins. This evolutionary conservatism suggests that almost all of their amino acids are important for the function of these histones. The N-terminus of these histones can be reversibly modified in the cell by acetylation of individual lysine residues, which removes the positive charge of lysines.

The nucleus is the region of the histone tail.

Beads on the A string

Short range of interaction

Linker histones

Fiber at 30 nm

Chromonema fiber

Long Range Fiber Interactions

nucleosome chromatin histone

The role of histones in DNA folding is important for the following reasons:

1) If chromosomes were just stretched DNA, it's hard to imagine how they could replicate and separate into daughter cells without getting tangled or broken.

2) In an extended state, the DNA double helix of each human chromosome would cross the cell nucleus thousands of times; thus, histones package a very long DNA molecule in an orderly manner into a nucleus several micrometers in diameter;

3) Not all DNA is folded in the same way, and the nature of the packaging of a region of the genome into chromatin probably affects the activity of the genes contained in this region.

In chromatin, DNA extends as a continuous double strand from one nucleosome to the next. Each nucleosome is separated from the next by a segment of linker DNA, which varies in size from 0 to 80 bp. On average, repetitive nucleosomes have a nucleotide interval of about 200 nucleotide pairs. In electron micrographs, this alternation of the histone octamer with coiled DNA and linker DNA gives the chromatin the appearance of "beads on a string" (after processing that unfolds the higher-order packaging).

Methylation how the covalent modification of histones is more complex than any other, since it can occur on both lysines and arginines. In addition, unlike any other modification in group 1, the consequences of methylation can be either positive or negative with respect to transcriptional expression, depending on the position of the residue in the histone (Table 10.1). Another level of complexity comes from the fact that there can be multiple methylated states for each residue. Lysines can be mono - (me1), di - (me2) or tri - (me3) methylated, while arginines can be mono - (me1) or di - (me2) methylated.

Phosphorylation RTM is best known because it has long been understood that kinases regulate signal transduction from the cell surface through the cytoplasm and into the nucleus, leading to changes in gene expression. Histones were among the first proteins to be phosphorylated. By 1991, it was discovered that when cells were stimulated to proliferate, so-called "immediate-early" genes were induced, and they became transcriptionally active and functioned to stimulate the cell cycle. This increased gene expression correlates with H3 histone phosphorylation (Mahadevan et al., 1991). H3 histone serine 10 (H3S10) has been shown to be an important phosphorylation site for transcription from yeast to humans and appears to be particularly important in Drosophila (Nowak and Corces, 2004)

Ubiquitination the process of attaching a "chain" of ubiquitin molecules to a protein (see Ubiquitin). At U. there is a connection of the C-terminus of ubiquitin with the side remains of lysine in a substrate. The polyubiquitin chain is hung at a strictly defined moment and is a signal indicating that this protein is subject to degradation.

Histone acetylation plays an important role in modulating chromatin structure during transcriptional activation, increasing chromatin accessibility to the transcriptional apparatus. It is believed that acetylated histones are less strongly bound to DNA and therefore it is easier for the transcription machine to overcome the resistance of chromatin packing. In particular, acetylation can facilitate the access and binding of transcription factors to their recognition elements on DNA. Enzymes that carry out the process of histone acetylation and deacetylation have now been identified, and we will probably soon learn more about how this is related to transcriptional activation.

It is known that acetylated histones are a sign of transcriptionally active chromatin.

Histones are the most biochemically studied proteins.

Organization of nucleosomes

The nucleosome is the basic unit of chromatin packaging. It consists of a DNA double helix wrapped around a specific complex of eight nucleosome histones (the histone octamer). The nucleosome is a disc-shaped particle with a diameter of about 11 nm, containing two copies of each of the nucleosomal histones (H2A, H2B, H3, H4). The histone octamer forms a protein core around which is double-stranded DNA (146 nucleotide pairs of DNA per histone octamer).

The nucleosomes that make up the fibrils are located more or less evenly along the DNA molecule at a distance of 10–20 nm from each other.

Data on the structure of nucleosomes were obtained using X-ray diffraction analysis of low and high definition nucleosome crystals, protein-DNA intermolecular crosslinks, and DNA cleavage within nucleosomes by nucleases or hydroxyl radicals. A. Klug built a model of the nucleosome, according to which DNA (146 bp) in the B-form (right-handed helix with a step of 10 bp) is wound on a histone octamer, in the central part of which the histones H3 and H4 are located, and on the periphery - H2a and H2b. The diameter of such a nucleosomal disk is 11 nm and its thickness is 5.5 nm. The structure consisting of a histone octamer and DNA wound around it is called the nucleosomal core particle. Core particles are separated from each other by linker DNA segments. The total length of the DNA segment included in the animal nucleosome is 200 (+/-15) bp.

Histone polypeptide chains contain several types of structural domains. The central globular domain and flexible protruding N- and C-terminal regions enriched in basic amino acids are called arms (arm). The C-terminal domains of polypeptide chains involved in histone-histone interactions within the core particle are predominantly in the form of an alpha helix with an extended central helical region, along which one shorter helix is laid on both sides. All known sites of reversible post-translational histone modifications that occur during the cell cycle or during cell differentiation are located in the flexible backbone domains of their polypeptide chains (Table I.2). At the same time, the N-terminal arms of H3 and H4 histones are the most conserved regions of the molecules, and histones as a whole are among the most evolutionarily conserved proteins. Via genetic research In the yeast S. cerevisiae, it was found that small deletions and point mutations in the N-terminal portions of histone genes are accompanied by profound and varied changes in the yeast cell phenotype, indicating the importance of histone molecular integrity in ensuring the proper functioning of eukaryotic genes. In solution, histones H3 and H4 can exist as stable tetramers (H3) 2 (H4) 2, while histones H2A and H2B can exist as stable dimers. A gradual increase in ionic strength in solutions containing native chromatin leads first to the release of H2A/H2B dimers and then H3/H4 tetramers.

Clarification fine structure nucleosomes in crystals was carried out by K. Luger et al. (1997) using high resolution X-ray diffraction analysis. It has been established that the convex surface of each histone heterodimer in the octamer is wrapped around by DNA segments 27-28 bp long, located at an angle of 140 degrees relative to each other, which are separated by linker regions 4 bp long.

Levels of DNA compaction: nucleosomes, fibrils, loops, mitotic chromosome

The first level of DNA compaction is the nucleosome. If chromatin is subjected to the action of nuclease, then it and DNA undergo decay into regularly repeating structures. After nuclease treatment, a fraction of particles is isolated from chromatin by centrifugation with a sedimentation rate of 11S. The 11S particles contain about 200 base pairs of DNA and eight histones. Such a complex nucleoprotein particle is called Nucleosomes. In it, histones form a protein core, on the surface of which DNA is located. DNA forms a site that is not associated with core proteins - a Linker, which, connecting two adjacent nucleosomes, passes into the DNA of the next nucleosome. They form "beads", globular formations of about 10 nm, sitting one after another on elongated DNA molecules. The second level of compaction is 30 nm fibril. The first, nucleosomal, level of chromatin compaction plays a regulatory and structural role, providing a DNA packing density of 6-7 times. In mitotic chromosomes and in interphase nuclei, chromatin fibrils with a diameter of 25-30 nm are detected. The solenoid type of nucleosome packing is distinguished: a thread of densely packed nucleosomes 10 nm in diameter forms coils with a helical pitch of about 10 nm. There are 6-7 nucleosomes per turn of such a superhelix. As a result of such packing, a helical-type fibril with a central cavity appears. Chromatin in the nuclei has a 25-nm fibril, which consists of contiguous globules of the same size - nucleomers. These nucleomeres are called superbeads ("superbids"). The main chromatin fibril, 25 nm in diameter, is a linear alternation of nucleomeres along a compacted DNA molecule. As part of the nucleomere, two turns of the nucleosomal fibril are formed, with 4 nucleosomes in each. The nucleomeric level of chromatin packing provides 40-fold compaction of DNA. Nuclesomal and nucleomeric (superbid) levels of chromatin DNA compaction are carried out by histone proteins. Loop domains of DNA-Tthird level structural organization of chromatin. V higher levels chromatin organization, specific proteins bind to specific regions of DNA, which forms large loops, or domains, at the binding sites. In some places there are clumps of condensed chromatin, rosette-shaped formations consisting of many loops of 30 nm fibrils, connected in a dense center. The average size of rosettes reaches 100-150 nm. Rosettes of chromatin fibrils-Chromomeres. Each chromomere consists of several loops containing nucleosomes, which are connected in one center. Chromomeres are connected to each other by regions of nucleosomal chromatin. Such a loop-domain structure of chromatin provides structural compaction of chromatin and organizes the functional units of chromosomes - replicons and transcribed genes.

Using the method of neutron scattering, it was possible to establish the shape and exact dimensions of nucleosomes; at a rough approximation, it is a flat cylinder or washer with a diameter of 11 nm and a height of 6 nm. Being located on a substrate for electron microscopy, they form "beads" - globular formations of about 10 nm, in single file, sitting in tandem on elongated DNA molecules. In fact, only the linker regions are elongated; the remaining three quarters of the DNA length are helically stacked along the periphery of the histone octamer. The histone octamer itself is thought to have a rugby-ball shape, comprising a (H3·H4) 2 tetramer and two independent H2A·H2B dimers. On fig. 60 shows the layout of histones in the core part of the nucleosome.

Composition of centromeres and telomeres

What are chromosomes, today almost everyone knows. These nuclear organelles, in which all genes are localized, constitute the karyotype of a given species. Under a microscope, chromosomes look like uniform, elongated dark rod-shaped structures, and the picture seen is unlikely to seem like an intriguing sight. Moreover, the preparations of the chromosomes of a great many living creatures that live on Earth differ only in the number of these rods and modifications of their shape. However, there are two properties that are common to chromosomes of all species.

Five stages of cell division (mitosis) are usually described. For simplicity, we will focus on three main stages in the behavior of the chromosomes of a dividing cell. At the first stage, there is a gradual linear contraction and thickening of chromosomes, then a cell division spindle is formed, consisting of microtubules. On the second, the chromosomes gradually move towards the center of the nucleus and line up along the equator, probably to facilitate the attachment of microtubules to the centromeres. In this case, the nuclear envelope disappears. At the last stage, the halves of the chromosomes - the chromatids - diverge. It seems that microtubules attached to the centromeres, like a tug, pull the chromatids to the poles of the cell. From the moment of divergence, the former sister chromatids are called daughter chromosomes. They reach the spindle poles and come together in parallel. The nuclear envelope is formed.

A model explaining the evolution of centromeres.

Up- centromeres (gray ovals) contain a specialized set of proteins (kinetochore), including histones CENH3 (H) and CENP-C (C), which in turn interact with spindle microtubules (red lines). In various taxa, one of these proteins evolves adaptively and in concert with the divergence of the primary centromere DNA structure.

At the bottom- changes in the primary structure or organization of centromeric DNA (dark gray oval) can create stronger centromeres, resulting in more microtubules attached.

Telomeres

The term "telomere" was proposed by G. Möller back in 1932. In his mind, it meant not only the physical end of the chromosome, but also the presence of a “terminal gene with special function sealing (sealing) of the chromosome”, which made it inaccessible to harmful effects (chromosomal rearrangements, deletions, nucleases, etc.). The presence of the terminal gene was not confirmed in subsequent studies, but the function of the telomere was determined accurately.

Later, another function was revealed. Since the usual mechanism of replication does not work at the ends of chromosomes, there is another way in the cell that maintains stable chromosome sizes during cell division. This role is performed by a special enzyme, telomerase, which acts like another enzyme, reverse transcriptase: it uses a single-stranded RNA template to synthesize the second strand and repair the ends of chromosomes. Thus, telomeres in all organisms perform two important tasks: they protect the ends of chromosomes and maintain their length and integrity.

A model of a protein complex of six telomere-specific proteins, which is formed on the telomeres of human chromosomes, is proposed. The DNA forms a t-loop, and the single-stranded protrusion is inserted into the double-stranded DNA region located distally (Fig. 6). The protein complex allows cells to distinguish between telomeres and chromosome break sites (DNA). Not all telomere proteins are part of the complex, which is redundant on telomeres but absent in other regions of the chromosomes. The protective properties of the complex stem from its ability to influence the structure of telomeric DNA in at least three ways: to determine the structure of the very tip of the telomere; participate in the formation of a t-loop; control the synthesis of telomeric DNA by telomerase. Related complexes have also been found on the telomeres of some other eukaryotic species.

Up -telomere at the time of chromosome replication, when its end is accessible to the telomerase complex, which carries out replication (doubling of the DNA chain at the very tip of the chromosome). After replication, telomeric DNA (black lines) together with proteins located on it (shown as multi-colored ovals) forms t - Petlyu (bottom of the picture ).

The time of DNA compaction in the cell cycle and the main factors stimulating processes

Recall the structure of chromosomes (from a biology course) - they are usually displayed as a pair of letters X, where each chromosome is a pair, and each has two identical parts - left and right chromatids. Such a set of chromosomes is typical for a cell that has already begun its division, i.e. cells that have undergone the process of DNA duplication. Doubling the amount of DNA is called the synthetic period, or S-period, of the cell cycle. They say that the number of chromosomes in a cell remains the same (2n), and the number of chromatids in each chromosome is doubled (4c - 4 chromatids per pair of chromosomes) - 2n4c. When dividing, one chromatid from each chromosome will enter the daughter cells and the cells will receive a complete diploid set of 2n2c.

The state of a cell (more precisely, its nucleus) between two divisions is called interphase. Three parts are distinguished in the interphase - the presynthetic, synthetic and postsynthetic periods.

Thus, the entire cell cycle consists of 4 time intervals: mitosis proper (M), presynthetic (G1), synthetic (S), and postsynthetic (G2) periods of interphase (Fig. 19). The letter G - from the English Gap - interval, gap. In the G1 period immediately after division, cells have a diploid DNA content per nucleus (2c). During the G1 period, cell growth begins mainly due to the accumulation of cellular proteins, which is determined by an increase in the amount of RNA per cell. During this period, the preparation of the cell for DNA synthesis (S-period) begins.

It was found that the suppression of protein or mRNA synthesis in the G1 period prevents the onset of the S period, since during the G1 period the synthesis of enzymes necessary for the formation of DNA precursors (for example, nucleotide phosphokinases), enzymes of RNA and protein metabolism occurs. This coincides with an increase in RNA and protein synthesis. This sharply increases the activity of enzymes involved in energy metabolism.

In the next, S-period, the amount of DNA per nucleus doubles and, accordingly, the number of chromosomes doubles. In different cells in the S-period, one can find different quantities DNA - from 2c to 4c. This is due to the fact that cells are examined at different stages of DNA synthesis (those that have just begun synthesis and those that have already completed it). The S-period is the nodal in the cell cycle. Not a single case of cells entering mitotic division is known without undergoing DNA synthesis.

The postsynthetic (G2) phase is also called premitotic. The last term emphasizes great importance to pass the next stage - the stage of mitotic division. In this phase, mRNA synthesis occurs, which is necessary for the passage of mitosis. Somewhat earlier than this, ribosome rRNA is synthesized, which determines cell division. Among the proteins synthesized at this time special place occupy tubulins - proteins of microtubules of the mitotic spindle.

At the end of the G2 period or during mitosis, as mitotic chromosomes condense, RNA synthesis drops sharply and completely stops during mitosis. Protein synthesis during mitosis decreases to 25% of the initial level and then in subsequent periods reaches its maximum in the G2 period, generally repeating the nature of RNA synthesis.

In the growing tissues of plants and animals there are always cells that are, as it were, outside the cycle. Such cells are usually called G0-period cells. It is these cells that are the so-called resting, temporarily or finally stopped reproducing cells. In some tissues, such cells can stay for a long time without especially changing their morphological properties: they retain, in principle, the ability to divide, turning into cambial, stem cells (for example, in hematopoietic tissue). More often, the loss (albeit temporary) of the ability to share is accompanied by the appearance of the ability to specialize, to differentiate. Such differentiating cells leave the cycle, but under special conditions they can re-enter the cycle. For example, most liver cells are in the G0 period; they do not participate in DNA synthesis and do not divide. However, when part of the liver is removed in experimental animals, many cells begin preparation for mitosis (G1-period), proceed to DNA synthesis, and can divide mitotically. In other cases, for example, in the epidermis of the skin, after leaving the cycle of reproduction and differentiation, the cells function for some time and then die (keratinized cells of the integumentary epithelium).

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The chemical composition of chromatin (chromosomes)

The study of the chemical organization of eukaryotic cell chromosomes has shown that they consist mainly of DNA and proteins that form a nucleoprotein complex.

DNA is the material carrier of the properties of heredity and variability and contains genetic information recorded using a special code. The amount of DNA in the nuclei of cells of an organism of a given species is constant and proportional to their ploidy. There is twice as much DNA in somatic cells of an organism as in gametes. A significant part of the substance of chromosomes is proteins, they account for about 65% of the mass.

All chromosomal proteins are divided into 2 groups: histones and nonhistone proteins. Histones are positively charged basic proteins that play a role in the packaging of chromosomal DNA and in the regulation of transcription. Histones are represented by 5 fractions: H1, H2A, H2B, H3, H4. In chromatin, all histone fractions are found in approximately equal amounts, except for H1, which is approximately two times less than any of the other fractions. The number of fractions of non-histone proteins exceeds 100; many of them are enzymes of RNA synthesis and processing, DNA replication and repair. They play a structural and regulatory role. Chromosome RNA is represented in part by transcription products that have not yet left the site of synthesis. Some RNA fractions have a regulatory function. Beyond DNA. proteins and RNA in the composition of chromosomes, lipids, polysaccharides, metal ions are found: Ca, Mg, Fe. Mass ratios are equal: DNA: histones: non-histone proteins: RNA: lipids (1: 1: (0.2-0.5) : (0.1-0.15): (0.01-0.03)). Other components are found in small quantities.

Chromatin- the main component of the cell nucleus; it is fairly easy to obtain from isolated interphase nuclei and from isolated mitotic chromosomes. To do this, use its property to go into a dissolved state during extraction with aqueous solutions with low ionic strength or simply deionized water.

Slide 3. There are two types of chromatin: heterochromatin and euchromatin. The first corresponds to the sections of chromosomes condensed during interphase, it is functionally inactive. This chromatin stains well; it is this chromatin that can be seen on the histological preparation. Heterochromatin is divided into structural (these are sections of chromosomes that are constantly condensed) and facultative (it can decondense and turn into euchromatin). Euchromatin corresponds to decondensation in interphase regions of chromosomes. This is a working, functionally active chromatin. It does not stain, it is not visible on the histological preparation. During mitosis, all euchromatin is condensed and incorporated into chromosomes.

Proteins are polymers, and amino acids are their monomer units.

Amino acids - these are organic compounds containing in their composition (in accordance with the name) an amino group NH2 and an organic acid, i.e. carboxyl, COOH group.

A protein molecule is formed as a result of the sequential connection of amino acids, while the carboxyl group of one acid interacts with the amino group of the neighboring molecule, as a result, a peptide bond is formed - CO-NH- and a water molecule is released. Slide 9

Chemical composition of histones. Features of physical properties and interaction with DNA

Histones- relatively small proteins with a very large proportion of positively charged amino acids (lysine and arginine); the positive charge helps the histones bind tightly to the DNA (which is highly negatively charged) regardless of its nucleotide sequence. The complex of both classes of proteins with the nuclear DNA of eukaryotic cells is called chromatin. Histones are a unique characteristic of eukaryotes and are present in vast numbers per cell (about 60 million molecules of each type per cell). Histone types fall into two main groups, nucleosomal histones and H1 histones, forming a family of highly conserved basic proteins, consisting of five large classes - H1 and H2A, H2B, H3 and H4. H1 histones are larger (about 220 amino acids) and have been found to be less conserved over the course of evolution. The size of histone polypeptide chains ranges from 220 (H1) to 102 (H4) amino acid residues. Histone H1 is highly enriched in Lys residues, histones H2A and H2B are characterized by a moderate content of Lys, the polypeptide chains of H3 and H4 histones are rich in Arg. Within each histone class (with the exception of H4), several subtypes of these proteins are distinguished based on amino acid sequences. This multiplicity is especially characteristic of mammalian H1 class histones. In this case, seven subtypes are distinguished, named H1.1-H1.5, H1o and H1t. Histones H3 and H4 are among the most conserved proteins. This evolutionary conservatism suggests that almost all of their amino acids are important for the function of these histones. The N-terminus of these histones can be reversibly modified in the cell by acetylation of individual lysine residues, which removes the positive charge of lysines.

The nucleus is the region of the histone tail.

Beads on the A string

Short range of interaction

Linker histones

Fiber at 30 nm

Chromonema fiber

Long Range Fiber Interactions

nucleosome chromatin histone

The role of histones in DNA folding is important for the following reasons:

1) If chromosomes were just stretched DNA, it's hard to imagine how they could replicate and separate into daughter cells without getting tangled or broken.
2) In an extended state, the DNA double helix of each human chromosome would cross the cell nucleus thousands of times; thus, histones package a very long DNA molecule in an orderly manner into a nucleus several micrometers in diameter;
3) Not all DNA is folded in the same way, and the nature of the packaging of a region of the genome into chromatin probably affects the activity of the genes contained in this region.

It is known that acetylated histones are a sign of transcriptionally active chromatin.

Histones are the most biochemically studied proteins.

Organization of nucleosomes

The nucleosomes that make up the fibrils are located more or less evenly along the DNA molecule at a distance of 10–20 nm from each other.

Data on the structure of nucleosomes were obtained using low- and high-resolution X-ray diffraction analysis of nucleosome crystals, protein-DNA intermolecular crosslinks, and DNA cleavage in nucleosomes using nucleases or hydroxyl radicals. A. Klug built a model of the nucleosome, according to which DNA (146 bp) in the B-form (right-handed helix with a step of 10 bp) is wound on a histone octamer, in the central part of which the histones H3 and H4 are located, and on the periphery - H2a and H2b. The diameter of such a nucleosomal disk is 11 nm and its thickness is 5.5 nm. The structure consisting of a histone octamer and DNA wound around it is called the nucleosomal core particle. Core particles are separated from each other by linker DNA segments. The total length of the DNA segment included in the animal nucleosome is 200 (+/-15) bp.

Histone polypeptide chains contain several types of structural domains. The central globular domain and flexible protruding N- and C-terminal regions enriched in basic amino acids are called arms (arm). The C-terminal domains of polypeptide chains involved in histone-histone interactions within the core particle are predominantly in the form of an alpha helix with an extended central helical region, along which one shorter helix is laid on both sides. All known sites of reversible post-translational histone modifications that occur during the cell cycle or during cell differentiation are located in the flexible backbone domains of their polypeptide chains (Table I.2). At the same time, the N-terminal arms of H3 and H4 histones are the most conserved regions of the molecules, and histones as a whole are among the most evolutionarily conserved proteins. Using genetic studies of the yeast S. cerevisiae, it was found that small deletions and point mutations in the N-terminal parts of histone genes are accompanied by profound and diverse changes in the phenotype of yeast cells, which indicates the importance of the integrity of histone molecules in ensuring the proper functioning of eukaryotic genes. In solution, histones H3 and H4 can exist as stable tetramers (H3) 2 (H4) 2, while histones H2A and H2B can exist as stable dimers. A gradual increase in ionic strength in solutions containing native chromatin leads first to the release of H2A/H2B dimers and then H3/H4 tetramers.

Refinement of the fine structure of nucleosomes in crystals was carried out by K. Luger et al. (1997) using high resolution X-ray diffraction analysis. It has been established that the convex surface of each histone heterodimer in the octamer is wrapped around by DNA segments 27-28 bp long, located at an angle of 140 degrees relative to each other, which are separated by linker regions 4 bp long.

Levels of DNA compaction: nucleosomes, fibrils, loops, mitotic chromosome

The first level of DNA compaction is the nucleosome. If chromatin is subjected to the action of nuclease, then it and DNA undergo decay into regularly repeating structures. After nuclease treatment, a fraction of particles is isolated from chromatin by centrifugation with a sedimentation rate of 11S. The 11S particles contain about 200 base pairs of DNA and eight histones. Such a complex nucleoprotein particle is called Nucleosomes. In it, histones form a protein core, on the surface of which DNA is located. DNA forms a site that is not associated with core proteins - a Linker, which, connecting two adjacent nucleosomes, passes into the DNA of the next nucleosome. They form "beads", globular formations of about 10 nm, sitting one after another on elongated DNA molecules. The second level of compaction is 30 nm fibril. The first, nucleosomal, level of chromatin compaction plays a regulatory and structural role, providing a DNA packing density of 6-7 times. In mitotic chromosomes and in interphase nuclei, chromatin fibrils with a diameter of 25-30 nm are detected. The solenoid type of nucleosome packing is distinguished: a thread of densely packed nucleosomes 10 nm in diameter forms coils with a helical pitch of about 10 nm. There are 6-7 nucleosomes per turn of such a superhelix. As a result of such packing, a helical-type fibril with a central cavity appears. Chromatin in the nuclei has a 25-nm fibril, which consists of contiguous globules of the same size - nucleomers. These nucleomeres are called superbeads ("superbids"). The main chromatin fibril, 25 nm in diameter, is a linear alternation of nucleomeres along a compacted DNA molecule. As part of the nucleomere, two turns of the nucleosomal fibril are formed, with 4 nucleosomes in each. The nucleomeric level of chromatin packing provides 40-fold compaction of DNA. Nuclesomal and nucleomeric (superbid) levels of chromatin DNA compaction are carried out by histone proteins. Loop domains of DNA-third level structural organization of chromatin. At higher levels of chromatin organization, specific proteins bind to specific regions of DNA, which form large loops, or domains, at the binding sites. In some places there are clumps of condensed chromatin, rosette-shaped formations consisting of many loops of 30 nm fibrils, connected in a dense center. The average size of rosettes reaches 100-150 nm. Rosettes of chromatin fibrils-Chromomeres. Each chromomere consists of several loops containing nucleosomes, which are connected in one center. Chromomeres are connected to each other by regions of nucleosomal chromatin. Such a loop-domain structure of chromatin provides structural compaction of chromatin and organizes the functional units of chromosomes - replicons and transcribed genes.

Composition of centromeres and telomeres

A model explaining the evolution of centromeres.

At the bottom- changes in the primary structure or organization of centromeric DNA (dark gray oval) can create stronger centromeres, resulting in more microtubules attached.

Telomeres

The term "telomere" was proposed by G. Möller back in 1932. In his view, it meant not only the physical end of the chromosome, but also the presence of a “terminal gene with a special function of sealing (sealing) the chromosome”, which made it inaccessible to harmful influences (chromosomal rearrangements, deletions, nucleases, etc.). The presence of the terminal gene was not confirmed in subsequent studies, but the function of the telomere was determined accurately.

Up -telomere at the time of chromosome replication, when its end is accessible to the telomerase complex, which carries out replication (duplication of the DNA chain at the very tip of the chromosome). After replication, telomeric DNA (black lines), together with proteins located on it (shown as multi-colored ovals), forms a t-loop ( bottom of the picture).

The time of DNA compaction in the cell cycle and the main factors stimulating processes

The state of a cell (more precisely, its nucleus) between two divisions is called interphase. Three parts are distinguished in the interphase - the presynthetic, synthetic and postsynthetic periods.

In the next, S-period, the amount of DNA per nucleus doubles and, accordingly, the number of chromosomes doubles. In different cells in the S-period, you can find different amounts of DNA - from 2c to 4c. This is due to the fact that cells are examined at different stages of DNA synthesis (those that have just begun synthesis and those that have already completed it). The S-period is the nodal in the cell cycle. Not a single case of cells entering mitotic division is known without undergoing DNA synthesis.

The postsynthetic (G2) phase is also called premitotic. The last term emphasizes its great importance for the passage of the next stage - the stage of mitotic division. In this phase, mRNA synthesis occurs, which is necessary for the passage of mitosis. Somewhat earlier than this, ribosome rRNA is synthesized, which determines cell division. Among the proteins synthesized at this time, a special place is occupied by tubulins - proteins of microtubules of the mitotic spindle.

Chromatin is a mass of genetic matter made up of DNA and proteins that condense to form chromosomes during eukaryotic division. Chromatin is found in our cells.

The main function of chromatin is to compress the DNA into a compact unit that is less bulky and can fit into the nucleus. Chromatin is made up of complexes of small proteins known as histones and DNA.

Histones help organize DNA into structures called nucleosomes, providing the foundation for DNA wrapping. The nucleosome is made up of a sequence of DNA strands that wrap around a set of eight histones called octomers. The nucleosome is further folded to form a chromatin fiber. Chromatin fibers coil and condense to form chromosomes. Chromatin enables a number of cellular processes including DNA replication, transcription, DNA repair, genetic recombination, and cell division.

Euchromatin and heterochromatin

Chromatin inside a cell can be compacted to varying degrees depending on the cell's stage in . The chromatin in the nucleus is contained in the form of euchromatin or heterochromatin. During interphase, the cell does not divide but undergoes a period of growth. Most of Chromatin is found in a less compact form known as euchromatin.

DNA is exposed to euchromatin, which allows for DNA replication and transcription. During transcription, the DNA double helix unwinds and opens up so that coding proteins can be copied. DNA replication and transcription are necessary for a cell to synthesize DNA, proteins, and in preparation for cell division (or).

A small percentage of chromatin exists as heterochromatin during interphase. This chromatin is densely packed, which does not allow gene transcription. Heterochromatin is stained with dyes in more dark color than euchromatin.

Chromatin in mitosis:

Prophase

During the prophase of mitosis, the chromatin fibers turn into chromosomes. Each replicated chromosome consists of two chromatids connected in .

metaphase

During metaphase, chromatin becomes extremely compressed. Chromosomes are aligned on the metaphase plate.

Anaphase

During anaphase, paired chromosomes () are separated and pulled out by the spindle microtubules to opposite poles of the cell.

Telophase

In telophase, each new one moves into its own nucleus. Chromatin fibers unwind and become less compacted. After cytokinesis, two genetically identical cells are formed. Each cell has the same number of chromosomes. Chromosomes continue to unwind and lengthen the forming chromatin.

Chromatin, chromosome and chromatid

People often have trouble distinguishing between the terms chromatin, chromosome, and chromatid. Although all three structures are made up of DNA and reside within the nucleus, each is defined separately.

Chromatin is made up of DNA and histones, which are packaged into thin fibers. These chromatin fibers do not condense, but can exist in either a compact form (heterochromatin) or a less compact form (euchromatin). Processes including DNA replication, transcription and recombination occur in euchromatin. During cell division, chromatin condenses to form chromosomes.

They are single-stranded structures of condensed chromatin. During the processes of cell division through mitosis and meiosis, chromosomes are replicated to ensure that each new daughter cell receives the correct number of chromosomes. The duplicated chromosome is double-stranded and has the familiar X shape. The two strands are identical and linked in a central region called the centromere.

It is one of two strands of replicated chromosomes. Chromatids connected by a centromere are called sister chromatids. At the end of cell division, sister chromatids separate from daughter chromosomes in newly formed daughter cells.

Chromatin structure and chemical composition. Structural organization of chromatin

Concept definition

Structural and functional components of chromatin

Histones and nucleosomes

Non-histone proteins

Levels of DNA compaction

Chromosomes

Euchromatin and heterochromatin

The biological role of chromatin

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