STRUCTURAL AND FUNCTIONAL ORGANIZATION OF GENETIC MATERIAL

4.2 Properties of DNA as a substance of heredity and variability

4.2.3 Changes in DNA nucleotide sequences.

4.2.4 Elementary units of variability of genetic material. Mouton. Recon

4.2.6 Mechanisms that reduce the adverse effect of gene mutations

4.3 Use of genetic information in life processes

4.3.2 Features of the organization and expression of genetic information in pro- and eukaryotes

1. Heredity and variability are the fundamental properties of the living

Life as a special phenomenon is characterized by the duration of existence in time (on Earth it originated more than 3.5 billion years ago), which is ensured by the continuity of generations of living systems. There is a change in the generations of cells in the body, a change in the generations of organisms in populations, a change in species in the biocenosis system, a change in the biocenoses that form the biosphere. The ability of living systems to self-reproduce lies at the basis of the continuous existence of life in time. The preservation of life in changing conditions is possible due to the evolution of living forms, during which they have changes that provide adaptation to a new environment. The continuity of existence and the historical development of living nature are due to two fundamental properties of life: heredity and variability.

In training courses, the properties of heredity and variability are traditionally considered in relation to the cell and the organism. In fact, they also manifest themselves at supraorganismal levels. At the cellular and organismal (ontogenetic) levels of organization of living things, heredity is understood as the property of cells or organisms in the process of self-reproduction to transfer to a new generation the ability to a certain type of metabolism and individual development, during which they form common features and properties of a given cell type and type of organisms, as well as some individual characteristics of parents. At the population-species level of life organization, heredity is manifested in maintaining a constant ratio of various genetic forms in a number of generations of organisms of a given population (species). At the biocenotic level, the continued existence of the biocenosis is ensured by the preservation of certain ratios of the species of organisms that form this biocenosis.

In the course of the emergence and development of life on Earth, heredity played a decisive role, as it consolidated biologically useful evolutionary acquisitions in a number of generations, ensuring a certain conservatism in the organization of living systems. Heredity is one of the main factors of evolution.

The continued existence of living nature in time against the background of changing conditions would be impossible if living systems did not have the ability to acquire and maintain certain changes that are useful in new environmental conditions. The property of living systems to acquire changes and exist in different variants is called variability.

In individual cells and organisms of the same species, variability, affecting their individual development, manifests itself in the appearance of differences between them. At the population-species level of life organization, this property is manifested in the presence of genetic differences between individual populations of a species, which underlies the formation of new species. The appearance of new species introduces changes in interspecific relationships in biocenoses. In a certain sense, variability reflects the dynamism of the organization of living systems and, along with heredity, is the leading factor in evolution. Despite the fact that, according to their results, heredity and variability are multidirectional, in living nature these two fundamental properties form an inseparable unity, which simultaneously ensures the preservation of existing biologically expedient qualities in the process of evolution and the emergence of new ones that make life possible in various conditions.

2. The history of the formation of ideas about the organization of the material substrate of heredity and variability

Heredity and variability, as the most important properties of any living system, are ensured by the functioning of a special material substrate. In the course of the historical development of biological science, ideas about its properties, organization and chemical nature are constantly expanding and becoming more complex.

In the 60s. 19th century the founder of genetics (the science of heredity and variability) G. Mendel (1865) made the first assumptions about the organization of hereditary material. Based on the results of his experiments on peas, he came to the conclusion that the hereditary material is discrete, i.e. represented by individual hereditary inclinations responsible for the development of certain characteristics of organisms. According to Mendel, in the hereditary material of sexually reproducing organisms, the development of a single trait is provided by a pair of allelic inclinations that came with germ cells from both parents. During the formation of gametes, only one of a pair of allelic inclinations enters each of them, therefore the gametes are always "pure". In 1909 V. Johansen called Mendel's "hereditary inclinations" genes.

80s 19th century were marked by important achievements in the field of cytology: mitosis and meiosis were described - the division of somatic and germ cells, respectively, during which nuclear structures - chromosomes - are naturally distributed between daughter cells (V. Voldeyer, 1888).

Data on the nature of the distribution of chromosomes in the process of cell division made it possible at the beginning of the 20th century. Boveri (1902-1907) and W. Setgon (1902-1903) conclude that the continuity of properties in a number of generations of cells and organisms is determined by the continuity of their chromosomes. Chromosomes began to be considered as material carriers of the hereditary program.

Further development of the chromosome theory of heredity, which combines ideas about hereditary inclinations and chromosomes, was carried out at the beginning of the 20th century. T. Morgan and his collaborators. In experiments performed on Drosophila, the previously stated assumption about the role of chromosomes in ensuring heredity was confirmed. It has been established that genes are located in chromosomes, located in them in a linear order. The genes of each chromosome form a linkage group, the number of which is determined by the number of chromosomes in the germ cells. Genes of the same linkage group are inherited, as a rule, together. However, in a number of cases, their recombination occurs due to crossing over, the frequency of which depends on the distance between the genes.

Thus, one of the most important principles of genetics, the unity of discreteness and continuity of hereditary material, was reflected in the chromosome theory.

It should be noted that also at the beginning of the XX century. facts were discovered that proved the presence in cells of extrachromosomal hereditary material located in various cytoplasmic structures and determining a special cytoplasmic heredity (K. Korrens, 1908).

Around the same time, X. de Vries (1901) laid the foundations for the theory of mutational variability associated with sudden changes in hereditary inclinations or chromosomes, which leads to changes in certain signs of the organism. In subsequent years, X-rays, radiation, certain chemicals and biological agents were found to have a mutagenic effect on chromosomes and genes.

As a result of these studies, it became obvious that heredity and variability are due to the functioning of the same material substrate.

In the first decades of the XX century. data were obtained that testify in favor of the dependence of the state of traits on the nature of the interaction of genes, which went beyond the relations of dominance and recessivity described by Mendel. From this came the idea of ​​the genetic apparatus as a system of interacting genes - the genotype, which is concentrated in the chromosome set - the karyotype.

The study of the chemical composition of chromosomes revealed two main types of compounds that form these structures - proteins and nucleic acids. In the first half of the XX century. researchers solved the question of the chemical nature of the substrate of heredity and variability. Initially, suggestions were made in favor of proteins. In 1928, F. Griffith set up an experiment on pneumococci, in which a change (transformation) of some hereditary properties of one bacterial strain was observed under the influence of material obtained from killed cells of another strain. The chemical nature of the substance that transforms the hereditary properties of bacteria was established only in 1944. O. Avery, who proved that it belongs to nucleic acids (DNA).

Other evidence for the involvement of DNA in ensuring heredity and variability are:

1) the constancy of the DNA content in all types of somatic cells of the body;

2) the correspondence of the DNA content to the ploidy of cells (in somatic cells it is twice as much as in germ cells, in polyploid cells it corresponds to the number of sets of chromosomes);

3) the phenomenon of genetic recombination in bacteria during their conjugation, during which a part of DNA penetrates from one cell into another and changes in the properties of the latter;

4) changing the hereditary properties of bacterial cells by transferring DNA from one strain to another with the help of a DNA phage - the phenomenon of transduction;

5) infectious activity of the isolated nucleic acid of viruses.

An important result of the purposeful study of nucleic acids was the creation by J. Watson and F. Crick (1953) of a spatial model of the DNA molecule.

In the second half of the XX century. the efforts of scientists are aimed at studying the properties of nucleic acids that form the basis of their genetic functions, ways of recording and reading hereditary information, the nature and structure of the genetic code, the mechanisms of regulation of gene activity in the process of formation of individual traits and the phenotype as a whole. In the 60s. the works of M. Nirenberg, S. Ochoa, X. Korana and others made a complete decoding of the genetic code, established the correspondence of nucleotide triplets in the nucleic acid molecule to certain amino acids. In the 70s. Genetic engineering methods began to be actively developed, which make it possible to purposefully change the hereditary properties of living organisms.

By the end of the 20th century, thanks to new molecular genetic technologies, it became possible to determine the nucleotide sequences in the DNA molecules of the genomes of various organisms (reading DNA texts). The DNA texts of the human genome, represented by a total of 3 billion base pairs, were mostly read by 2001. The scientific and practical direction of molecular biology, which aims to determine the nucleotide sequences of DNA molecules, is called genomics.

3. General properties of the genetic material and levels of organization of the genetic apparatus

Based on the above definitions of heredity and variability, we can assume what requirements the material substrate of these two properties of life must meet.

First, the genetic material must be capable of self-replication in order to in the process of reproduction, transmit hereditary information, on the basis of which the formation of a new generation will be carried out. Secondly, in order to ensure the stability of characteristics in a number of generations, the hereditary material must maintain its organization constant. Thirdly, the material of heredity and variability must be capable of acquiring changes and reproducing them, making it possible for the historical development of living matter in changing conditions. Only if it meets the specified requirements, the material substrate of heredity and variability can ensure the duration and continuity of the existence of living nature and its evolution.

Modern ideas about the nature of the genetic apparatus make it possible to distinguish three levels of its organization: gene, chromosomal and genomic. On each of them, the main properties of the material of heredity and variability and certain patterns of its transmission and functioning are manifested.

4. Gene level of organization of the genetic apparatus

The elementary functional unit of the genetic apparatus, which determines the possibility of developing a particular trait of a cell or organism of a given species, is a gene (hereditary deposit, according to G. Mendel). By transferring genes in a series of generations of cells or organisms, material continuity is achieved - the inheritance of parental traits by descendants.

A sign is understood as a unit of morphological, physiological, biochemical, immunological, clinical and any other discreteness of organisms (cells), i.e. a separate quality or property by which they differ from each other.

Most of the features of organisms or cells listed above belong to the category of complex features, the formation of which requires the synthesis of many substances, primarily proteins with specific properties - enzymes, immunoproteins, structural, contractile, transport and other proteins. The properties of a protein molecule are determined by the amino acid sequence of its polypeptide chain, which is directly specified by the sequence of nucleotides in the DNA of the corresponding gene and is an elementary or simple feature.

The main properties of a gene as a functional unit of the genetic apparatus are determined by its chemical organization,

4.1 Chemical organization of the gene

Studies aimed at elucidating the chemical nature of hereditary material have irrefutably proved that the material substrate of heredity and variability are nucleic acids, which were discovered by F. Miescher (1868) in the nuclei of pus cells. Nucleic acids are macromolecules, i.e. have a high molecular weight. These are polymers consisting of monomers - nucleotides, including three components: sugar (pentose), phosphate and a nitrogenous base (purine or pyrimidine). A nitrogenous base (adenine, guanine, cytosine, thymine or uracil) is attached to the first carbon atom in the C-1 pentose molecule, and a phosphate is attached to the fifth carbon atom C-5 "using an ether bond; the third carbon atom C-3 "always has a hydroxyl group - OH (Fig. 1).

The connection of nucleotides into a nucleic acid macromolecule occurs by the interaction of the phosphate of one nucleotide with the hydroxyl of another so that a phosphodiester bond is established between them (Fig. 2). The result is a polynucleotide chain. The backbone of the chain consists of alternating phosphate and sugar molecules. One of the nitrogenous bases listed above is attached to the pentose molecules in the C-1 "position (Fig. 3).

Fig.1. Diagram of the nucleotide structure

See text for explanation; the nucleotide component designations used in this figure are retained in all subsequent nucleic acid schemes

The assembly of the polynucleotide chain is carried out with the participation of the polymerase enzyme, which ensures the attachment of the phosphate group of the next nucleotide to the hydroxyl group in position 3 "of the previous nucleotide (Fig. 3.3). Due to the noted specificity of the action of the named enzyme, the growth of the polynucleotide chain occurs only at one end: there where the free hydroxyl is in position 3". The beginning of the chain always carries a phosphate group in position 5 ". This allows you to select the 5" and 3" ends in it.

Among nucleic acids, two types of compounds are distinguished: deoxyribonucleic (DNA) and ribonucleic (RNA) acids. The study of the composition of the main carriers of hereditary material - chromosomes - found that their most chemically stable component is DNA, which is the substrate of heredity and variability.

4.1.1 Structure of DNA. Model by J. Watson and F. Crick

DNA consists of nucleotides, which include sugar - deoxyribose, phosphate and one of the nitrogenous bases - purine (adenine or guanine) or pyrimidine (thymine or cytosine). A feature of the structural organization of DNA is that its molecules include two polynucleotide chains interconnected in a certain way. In accordance with the three-dimensional DNA model proposed in 1953 by the American biophysicist J. Watson and the English biophysicist and geneticist F. Crick, these chains are connected to each other by hydrogen bonds between their nitrogenous bases according to the principle of complementarity. Adenine of one chain is connected by two hydrogen bonds with thymine of another chain, and three hydrogen bonds are formed between guanine and cytosine of different chains. Such a connection of nitrogenous bases provides a strong connection between the two chains and maintaining an equal distance between them throughout.

Fig.4. Diagram of the structure of the DNA molecule. The arrows indicate the anti-parallelism of the targets.

Another important feature of the association of two polynucleotide chains in a DNA molecule is their antiparallelism: the 5" end of one chain is connected to the 3" end of the other, and vice versa (Fig. 4).

X-ray diffraction data showed that a DNA molecule consisting of two strands forms a helix twisted around its own axis. The helix diameter is 2 nm, the pitch length is 3.4 nm. Each turn contains 10 pairs of nucleotides.

Most often, double helixes are right-handed - when moving up along the axis of the helix, the chains turn to the right. Most DNA molecules in solution are in the right-handed - B-form (B-DNA). However, there are also left-handed forms (Z-DNA). How much of this DNA is present in cells and what is its biological significance has not yet been established (Fig. 3.5).

Fig.5. Spatial models of left-handed Z-shape (I) and right-handed B-shape (II) of DNA

Thus, in the structural organization of the DNA molecule, one can distinguish the primary structure - a polynucleotide chain, the secondary structure - two complementary and antiparallel polynucleotide chains connected by hydrogen bonds, and the tertiary structure - a three-dimensional helix with the above spatial characteristics.

4.1.2 A method of recording genetic information in a DNA molecule. Biological code and its properties

Primarily, all the diversity of life is determined by the diversity of protein molecules that perform various biological functions in cells. The structure of proteins is determined by the set and order of amino acids in their peptide chains. It is this sequence of amino acids in peptides that is encrypted in DNA molecules using a biological (genetic) code. The relative primitiveness of the DNA structure, representing the alternation of only four different nucleotides, for a long time prevented researchers from considering this compound as a material substrate of heredity and variability, in which extremely diverse information should be encrypted.

In 1954 G. Gamow suggested that the encoding of information in DNA molecules should be carried out by combinations of several nucleotides. In the variety of proteins that exist in nature, about 20 different amino acids have been found. To encrypt such a number of them, only a triplet code can provide a sufficient number of combinations of nucleotides, in which each amino acid is encrypted by three adjacent nucleotides. In this case, 4 3 = 64 triplets are formed from four nucleotides. A code consisting of two nucleotides would make it possible to encode only 4 2 = 16 different amino acids.

The complete decoding of the genetic code was carried out in the 60s. our century. Of the 64 possible DNA triplets, 61 encode different amino acids; the remaining 3 are called meaningless, or "nonsense triplets". They do not encode amino acids and act as punctuation marks when reading hereditary information. These include ATT, ACT, ATC.

Attention is drawn to the obvious redundancy of the code, which is manifested in the fact that many amino acids are encrypted by several triplets (Fig. 6). This property of the triplet code, called degeneracy, is very important, since the occurrence of changes in the structure of the DNA molecule by the type of replacement of one nucleotide in the polynucleotide chain may not change the meaning of the triplet. The resulting new combination of three nucleotides encodes the same amino acid.

In the process of studying the properties of the genetic code, its specificity was discovered. Each triplet can code for only one specific amino acid. An interesting fact is the complete correspondence of the code in various types of living organisms. Such universality of the genetic code testifies to the unity of the origin of all the diversity of living forms on Earth in the process of biological evolution. Minor differences in the genetic code are found in the DNA of mitochondria of some species. This does not generally contradict the statement about the universality of the code, but it testifies in favor of a certain divergence in its evolution in the early stages of the existence of life. Deciphering the code in the DNA of mitochondria of various species showed that in all cases, mitochondrial DNA has a common feature: the ACT triplet is read as ACC, and therefore it turns from a nonsense triplet into the tryptophan amino acid cipher.

Fig.6. Amino acids and DNA triplets encoding them

Other features are specific to different species of organisms. In yeast, the GAT triplet, and possibly the entire GA family, encodes threonine instead of the amino acid leucine. In mammals, the TAG triplet has the same meaning as TAC and codes for the amino acid methionine instead of isoleucine. Triplets of TCH and TCC in the DNA of mitochondria of some species do not encode amino acids, being nonsense triplets. Along with tripletity, degeneracy, specificity and universality, the most important characteristics of the genetic code are its continuity and non-overlapping of codons during reading. This means that the nucleotide sequence is read triple by triplet without gaps, while neighboring triplets do not overlap each other, i.e. each individual nucleotide is part of only one triplet for a given reading frame (Fig. 3.7). The proof of the non-overlapping of the genetic code is the replacement of only one amino acid in the peptide when replacing one nucleotide in DNA. In the case of inclusion of a nucleotide in several overlapping triplets, its replacement would entail the replacement of 2-3 amino acids in the peptide chain.

Fig.7. Continuity and indisputability of the genetic code when reading hereditary information.

Nucleotides are nucleotides.

4.2 Properties of DNA as a substance of heredity and variability

4.2.1 Self-reproduction of hereditary material. DNA replication

One of the main properties of the material of heredity is its ability to copy itself - replication. This property is provided by the peculiarities of the chemical organization of the DNA molecule, which consists of two complementary strands. In the process of replication, a complementary chain is synthesized on each polynucleotide chain of the parent DNA molecule. As a result, two identical double helixes are formed from one DNA double helix. Such a method of doubling molecules, in which each daughter molecule contains one parent and one newly synthesized chain, is called semi-conservative.

For replication to take place, the parent DNA strands must be separated from each other to become templates on which complementary strands of daughter molecules will be synthesized.

The initiation of replication is carried out in special regions of DNA, designated ori (from the English origin - the beginning). They include a 300 bp sequence recognized by specific proteins. The DNA double helix in these loci is divided into two strands, and, as a rule, on both sides of the origin of replication, areas of divergence of polynucleotide chains are formed - replication forks that move in opposite directions from the ori locus. Between the replication forks, a structure called the replication eye is formed, where new polynucleotide chains are formed on two strands of maternal DNA (Fig. 8, A).

With the help of the enzyme helicase, which breaks hydrogen bonds, the double helix of DNA unwinds at the points of origin of replication. The resulting single DNA strands are bound by special destabilizing proteins that stretch the backbones of the chains, making their nitrogenous bases available for binding to complementary nucleotides located in the nucleoplasm. On each of the chains formed in the region of the replication fork, with the participation of the enzyme DNA polymerase, the synthesis of complementary chains is carried out (Fig. 8, B).

Fig.8. Replication start area. replication fork

A. Formation of a replication eye.

B. Region of the replication fork in the DNA molecule

During synthesis, the replication forks move along the parent helix in opposite directions, capturing new zones.

Separation of helical strands of parental DNA by the enzyme helicase causes the appearance of supercoils in front of the replication fork. This is explained by the fact that for every 10 pairs of nucleotides that form one turn of the helix, the parent DNA must complete one complete turn around its axis. Therefore, in order to advance the replication fork, the entire DNA molecule in front of it would have to rotate rapidly, which would require a large expenditure of energy. This is not actually observed due to a special class of proteins called DNA topoisomerases. Topoisomerase breaks one of the DNA strands, allowing it to revolve around the second strand. This weakens the accumulated tension in the DNA double helix (Fig. 9).

Free nucleotides from the nucleoplasm, where they are present in the form of deoxyribonucleoside gryphosphates: dATP, dGTP, dCTP, dTTP, join the released hydrogen bonds of the nucleotide sequences of the separated parental chains. Complementary nucleoside triphosphate forms hydrogen bonds with a specific base of the parent DNA strand. Then, with the participation of the DNA polymerase enzyme, it binds by a phosphodiester bond to the previous nucleotide of the newly synthesized chain, while giving inorganic pyrophosphate (Fig. 10).

As DNA polymerase adds the next nucleotide to the OH group at the 3' position of the preceding nucleotide, the chain gradually lengthens at its 3' end.

A feature of DNA polymerase is its inability to start the synthesis of a new polynucleotide chain by simply linking two nucleoside triphosphates: the 3 "-OH-terminus of any polynucleotide chain paired with the template DNA chain is required, to which DNA polymerase can only add new nucleotides. Such a polynucleotide The leotide chain is called the seed or primer.

The role of a primer for the synthesis of DNA polynucleotide chains during replication is performed by short RNA sequences formed with the participation of the RNA primase enzyme (Fig. 11). This feature of DNA polymerase means that only a DNA chain carrying a paired primer, which has a free 3'-OH-end, can serve as a template for replication.

Fig.9. Breaking one of the DNA chains with the help of the enzyme DNA topoisomerase: I - DNA topoisomerase forms a covalent bond with one of the phosphate groups of DNA (upper chain); II - as a result of breaking the phosphodiester bond in one polynucleotide chain around the corresponding bond of the other chain, rotation occurs, which relieves the tension caused by the divergence of two DNA chains in the area of ​​the replication fork; III - after the release of tension in the DNA helix, spontaneous separation of DNA topoisomerase and restoration of the phosphodiester bond in the DNA chain occurs

The ability of DNA polymerase to assemble a polynucleotide in the direction from the 5" to the 3" end when two DNA strands are joined antiparallel means that the replication process should proceed differently on them. Indeed, if on one of the matrices (3" → 5") the assembly of a new chain occurs continuously from the 5" to the 3" end and it gradually lengthens at the 3" end, then the other chain synthesized on the matrix (5" → 3 "), should grow from the 3" to the 5" end. This is contrary to the direction of action of the DNA polymerase enzyme.

Fig.10. Attachment of the next nucleotide to the daughter strand of DNA synthesized with the participation of DNA polymerase: FF-pyrophosphate

It has now been established that the synthesis of the second strand of DNA is carried out by short fragments (Okazaki fragments) also in the direction from the 5" to the 3" end (by the type of sewing "backward with a needle"). In prokaryotes, Okazaki fragments contain from 1000 to 2000 nucleotides, in eukaryotes they are much shorter (from 100 to 200 nucleotides). The synthesis of each such fragment is preceded by the formation of an RNA primer about 10 nucleotides long. The newly formed fragment is connected with the previous fragment with the help of the DNA ligase enzyme after the removal of its RNA primer (Fig. 12, A).

Due to these features, the replication fork is asymmetric. Of the two synthesized daughter chains, one is built continuously, its synthesis is faster, and this chain is called the leader. Synthesis of the other chain is slower, as it is assembled from separate fragments that require the formation and then removal of the RNA primer. Therefore, such a chain is called lagging (lagging). Although individual fragments are formed in the direction 5 "→ 3", in general, this chain grows in the direction 3 "→ 5" (Fig. 3.12, A). In view of the fact that two replication forks usually start from the ori locus, going in opposite directions, the synthesis of leading strands in them occurs on different strands of maternal DNA (Fig. 12, B). The end result of the replication process is the formation of two DNA molecules whose nucleotide sequence is identical to that of the parent DNA double helix.

Fig.11. Reaction scheme for the synthesis of a short RNA primer catalyzed by RNA primase

The considered sequence of events occurring in the course of replicative synthesis suggests the participation of a whole system of enzymes: helicase, topoisomerase, destabilizing proteins, DNA polymerase, and others, acting together in the area of ​​the replication fork (Fig. 13).

DNA replication in pro- and eukaryotes is basically similar, however, the rate of synthesis in eukaryotes (about 100 nucleotides/s) is an order of magnitude lower than in prokaryotes (1000 nucleotides/s). The reason for this may be the formation of eukaryotic DNA of sufficiently strong bonds with proteins, which hinders its despiralization, which is necessary for replicative synthesis.

A DNA fragment from the point of origin of replication to the point of its termination forms a unit of replication - the replicon. Once started at the point of origin (the on locus), replication continues until the entire replicon has been duplicated. The circular DNA molecules of prokaryotic cells have one on locus and are entirely separate replicons. Eukaryotic chromosomes contain a large number of replicons. In this regard, the duplication of the DNA molecule located along the eukaryotic chromosome begins at several points. In different replicons, doubling can occur at different times or simultaneously.

Rice. 12. Synthesis of two daughter strands of DNA on different strands of the parent molecule

A. Due to the antiparallelism of DNA strands, the synthesis of daughter strands proceeds differently; on the upper parent strand, a continuously leading strand is synthesized; on the lower parent strand, the daughter strand is assembled from Okazaki fragments - a lagging strand.

B. Synthesis of leading strands in multidirectional forks occurs on different strands of maternal DNA

4.2.2 Mechanisms for maintaining the nucleoside DNA sequence. Chemical stability. Replication. Repair

To maintain the main characteristics of a cell or organism throughout their life, as well as in a number of generations, the hereditary material must be resistant to external influences or there must be mechanisms for correcting the changes that occur in it. In nature, both factors are used. The third factor is the accuracy of copying the nucleotide sequences of maternal DNA during its replication.

Fig.13. Proteins involved in the process of DNA replication

DNA helicase unwinds the DNA double helix, separating its polynucleotide chains; destabilizing proteins straighten a portion of the DNA chain; DNA topoisomerase breaks the phosphodiester bond in one of the polycarbonate DNA strands, relieving the tension caused by helix unwinding and strand separation at the replication fork; RNA primase synthesizes RNA primers for the daughter strand and for each Okazaki fragment; DNA polymerase performs continuous synthesis of the leading strand and synthesis of Okazaki fragments of the lagging strand; DNA ligase ligates Okazaki fragments after removal of RNA primer

In terms of reactivity, DNA molecules are classified as chemically inert substances. It is known that the role of the substance of heredity can be performed not only by DNA, but also by RNA (some viruses). It is believed that the choice in favor of DNA is due to its lower reactivity compared to RNA.

The mechanism of replication discussed above is characterized by an extremely high fidelity in the reproduction of the DNA structure. When duplicating DNA, errors occur on average with a frequency of 1·10 -6 complementary base pairs.

In maintaining high replication fidelity, an important role belongs primarily to the enzyme DNA polymerase. This enzyme selects the necessary nucleotides from among the nucleoside triphosphates (ATP, TTP, GTP, CTP) present in the nuclear sap, accurately attaches them to the DNA template chain and includes them in the growing daughter chain. The frequency of inclusion of incorrect nucleotides at this stage is 1·10 -5 base pairs.

Such errors in the work of DNA polymerase are associated with the appearance of altered forms of nitrogenous bases, which form "illegal" pairs with the bases of the parent chain. For example, an altered form of cytosine instead of guanine is hydrogen bonded to adenine. As a result, an erroneous nucleotide is included in the growing DNA chain. The rapid transition of the altered form of such a base into the usual one disrupts its binding to the template, an unpaired 3 "-OH-end of the growing DNA chain appears. In this situation, the self-correction mechanism is activated, carried out by DNA polymerase (or an enzyme closely related to it - editing endonuclease). Self-correction consists in the cleavage of a nucleotide erroneously included in the DNA chain that is not paired with the template (Fig. 14).The consequence of self-correction is a decrease in the error rate by 10 times (from 10 -5 to 10 -6).

Despite the effectiveness of self-correction, errors are detected during replication after DNA duplication. This is especially often observed when the concentration of four nucleoside triphosphates in the surrounding substrate is disturbed. A significant part of the changes also occurs in DNA molecules as a result of spontaneous processes associated with the loss of purine bases - adenine and guanine (apurinization) - or the deamination of cytosine, which turns into uracil. The frequency of the last changes reaches 100 per 1 genome/day.

The bases contained in DNA can change under the influence of reactive compounds that disrupt their normal pairing, as well as under the action of ultraviolet radiation, which can cause the formation of a covalent bond between two adjacent thymine residues in DNA (thymine dimers). These changes in the next cycle of replication should lead either to the loss of base pairs in the daughter DNA, or to the replacement of some pairs with others. These changes do accompany each cycle of DNA replication, but their frequency is much less than it should be. This is explained by the fact that most changes of this kind are eliminated due to the action of the mechanism of repair (molecular restoration) of the original DNA nucleotide sequence.

The repair mechanism is based on the presence of two complementary chains in the DNA molecule. The distortion of the nucleotide sequence in one of them is detected by specific enzymes. Then the corresponding site is removed and replaced by a new one, synthesized on the second complementary DNA strand. Such repair is called excisional, i.e. with "cutting" (Fig. 15). It is carried out before the next replication cycle, so it is also called pre-replicative.

Fig.14. Scheme of the correction process during DNA synthesis:

I-inclusion in the DNA chain of a nucleotide with a modified (tautomeric) form of cytoein, which "illegally" pairs with adenine; II - the rapid transition of cytosine to its normal form disrupts its pairing with adenine; the unpaired 3"-OH-end of the synthesized chain prevents its further elongation under the action of DNA polymerase; III - DNA polymerase removes the illegal nucleotide, as a result of which the 3"-OH-end paired with the template reappears; IV - DNA polymerase continues to extend the chain at the 3'-OH-end.

Restoration of the original DNA structure requires the participation of a number of enzymes. An important point in starting the repair mechanism is the detection of an error in the DNA structure. Often such errors occur in the newly synthesized strand during replication. Repair enzymes must detect exactly this chain. In many species of living organisms, the newly synthesized DNA chain differs from the maternal degree of methylation of its nitrogenous bases, which lags behind synthesis. In this case, the unmethylated chain undergoes repair. The object of recognition by repair enzymes can also be breaks in the DNA chain. In higher organisms, where DNA synthesis does not occur continuously, but by individual replicons, the newly synthesized DNA chain has breaks, which makes it possible to recognize it. Restoration of the DNA structure in the event of the loss of purine bases of one of its chains involves the detection of a defect using the enzyme endonuclease, which breaks the phosphoester bond at the site of damage to the chain. Then the altered site with several nucleotides adjacent to it is removed by the exonuclease enzyme, and in its place, in accordance with the order of the bases of the complementary chain, the correct nucleotide sequence is formed (Fig. 15).

Fig.15. Scheme of excisional, pre-replicative DNA repair.

When one of the bases in the DNA chain changes, around 20 DNA glycosylase enzymes take part in the restoration of the original structure. They specifically recognize damage caused by deamination, alkylation, and other structural transformations of bases. Such modified bases are removed. There are areas devoid of bases, which are repaired, as with the loss of purines. If the restoration of the normal structure is not carried out, for example, in the case of deamination of nitrogenous bases, some pairs of complementary bases are replaced by others - the C-G pair can be replaced by a T-A pair, etc. .

The formation of thymine dimers (T-T) in polynucleotide chains under the action of UV rays requires the participation of enzymes that recognize not individual altered bases, but more extensive damage to the DNA structure. The reparative process in this case is also associated with the removal of the site carrying the dimer and the restoration of the normal nucleotide sequence by synthesis on the complementary DNA strand.

In the event that the excision repair system does not correct a change that has arisen in one DNA strand, this change is fixed during replication and it becomes the property of both DNA strands. This leads to the replacement of one pair of complementary nucleotides with another or to the appearance of breaks (gaps) in the newly synthesized chain against the altered regions. Restoration of the normal DNA structure can also occur after replication.

Postreplicative repair is carried out by recombination (exchange of fragments) between two newly formed DNA double helixes. An example of such post-replicative repair is the restoration of the normal DNA structure when thymine dimers (T-T) appear, when they are not eliminated spontaneously under the action of visible light (light repair) or during pre-replicative excisional repair.

Covalent bonds that occur between adjacent thymine residues make them incapable of binding to complementary nucleotides. As a result, breaks (gaps) that are recognized by repair enzymes appear in the newly synthesized DNA strand. Restoration of the integrity of the new polynucleotide chain of one of the daughter DNAs is carried out due to recombination with the corresponding normal maternal chain of the other daughter DNA. The gap formed in the parent chain is then filled by synthesis on the complementary polynucleotide chain (Fig. 16). The often observed exchange of material between sister chromatids (Fig. 17) can be considered as a manifestation of such post-replicative repair, carried out by recombination between the chains of two daughter DNA molecules.

Fig.16. Diagram of post-replicative DNA repair:

I - occurrence of thymine dimer in one of the DNA strands;

II - the formation of a "gap" in the newly synthesized strand against the altered region of the parent molecule after replication (the arrow indicates the subsequent filling of the "gap" with a region from the corresponding strand of the second daughter DNA molecule);

III - restoration of the integrity of the daughter chain of the upper molecule due to recombination and in the lower molecule due to synthesis on the complementary chain

Fig.17. Interchromatid exchanges (indicated by arrows)

In the course of pre-replicative and post-replicative repair, most of the damage to the DNA structure is restored. However, if too much damage occurs in the hereditary material of the cell and some of them are not eliminated, the system of inducible (excited) repair enzymes (SOS-system) is turned on. These enzymes fill gaps by restoring the integrity of the synthesized polynucleotide chains without strictly observing the principle of complementarity. That is why sometimes the repair processes themselves can serve as a source of persistent changes in the DNA structure (mutations). The named reaction also applies to the SOS system.

If in the cell, despite the ongoing repair, the amount of damage to the DNA structure remains high, the processes of DNA replication are blocked in it. Such a cell does not divide, which means that it does not transmit the changes that have arisen to offspring.

The arrest of the cell cycle caused by DNA damage, combined with the impossibility of molecular repair of the altered hereditary material, can, with the participation of a protein whose synthesis is controlled by the p53 gene, lead to the activation of the process of self-destruction (apoptosis) of a defective cell in order to eliminate it from the body.

Thus, an extensive set of various repair enzymes performs a continuous "examination" of DNA, removing damaged areas from it and helping to maintain the stability of the hereditary material. The joint action of replication enzymes (DNA polymerase and editing endonuclease) and repair enzymes ensures a fairly low error rate in DNA molecules, which is maintained at the level of 1 × 10 -9 pairs of altered nucleotides per genome. With a human genome size of 3 × 10 9 base pairs, this means about 3 errors per replicating genome. At the same time, even this level is sufficient for the formation of significant genetic diversity in the form of gene mutations during the existence of life on Earth.

4.2.3 Changes in DNA nucleotide sequences.

Uncorrected changes in the chemical structure of genes, reproduced in successive cycles of replication and manifested in offspring in the form of new variants of traits, are called gene mutations.

Changes in the DNA structure that makes up a gene can be divided into three groups. Mutations of the first group consist in the replacement of some bases by others. They make up about 20% of spontaneously occurring gene changes. The second group of mutations is caused by a frame shift that occurs when the number of nucleotide pairs in the gene is changed. Finally, the third group is represented by mutations associated with a change in the order of nucleotide sequences within a gene (inversion).

Mutations according to the type of replacement of nitrogenous bases. These mutations occur for a number of specific reasons. One of them may be a change in the structure of a base that is already included in the DNA helix, which occurs by chance or under the influence of specific chemical agents. If such an altered form of the base remains unnoticed by the repair enzymes, then during the next replication cycle it can attach another nucleotide to itself. An example is the deamination of cytosine, which turns into uracil spontaneously or under the influence of nitrous acid (Fig. 18). The resulting uracil, not noticed by the enzyme DNA glycosylase, during replication combines with adenine, which subsequently attaches the thymidyl nucleotide. As a result, the C-G pair is replaced in DNA by the T-A pair (Fig. 19, I). Deamination of methylated cytosine converts it to thymine (see Figure 3.18). The thymidyl nucleotide, being a natural component of DNA, is not detected as a change by repair enzymes and adds an adenyl nucleotide during the next replication. As a result, instead of the C-G pair, the T-A pair also appears in the DNA molecule (Fig. 19, II).

Fig.18. Spontaneous deamination of cytosine

Another reason for the substitution of bases may be the erroneous inclusion in the synthesized DNA chain of a nucleotide carrying a chemically modified form of the base or its analogue. If this error remains unnoticed by the replication and repair enzymes, the changed base is included in the replication process, which often leads to the replacement of one pair with another. An example of this is the addition of a nucleotide with 5-bromouracil (5-BU), similar to the thymidyl nucleotide, to the adenine of the maternal chain during replication. During subsequent replication, 5-BU more readily attaches to itself not adenine, but guanine. Guanine in the course of further doubling forms a complementary pair with cytosine. As a result, the A-T pair is replaced in the DNA molecule by the G-C pair (Fig. 20).

Rice. 19. Mutations by the type of base substitution (deamination of nitrogenous bases in the DNA chain):

I - conversion of cytosine to uracil, replacement of the C-G-pair with a T-A-pair;

II - conversion of methyl-cytosine to thymine, replacement of the C-G-pair with a T-A-pair

From the above examples, it can be seen that changes in the structure of the DNA molecule by the type of base substitution occur either before or during replication, initially in one polynucleotide chain. If such changes are not corrected during repair, then during subsequent replication they become the property of both DNA strands.

Rice. 20. Mutations by the type of base substitution (inclusion of a nitrogenous base analog in DNA replication)

The consequence of replacing one pair of complementary nucleotides with another is the formation of a new triplet in the DNA nucleotide sequence encoding the amino acid sequence in the peptide chain. This may not affect the structure of the peptide if the new triplet is "synonymous" with the previous one, i.e. will code for the same amino acid. For example, the amino acid valine is encrypted with four triplets: CAA, CAG, CAT, CAC. Replacing the third base in any of these triplets will not change its meaning (the degeneracy of the genetic code).

In the case when the newly emerged triplet encodes another amino acid, the structure of the peptide chain and the properties of the corresponding protein change. Depending on the nature and place of the replacement, the specific properties of the protein change to varying degrees. Cases are known when the replacement of only one amino acid in a peptide significantly affects the properties of the protein, which manifests itself in a change in more complex features. An example is the change in the properties of human hemoglobin in sickle cell anemia (Fig. 21). In such hemoglobin- (HbS) (unlike normal HbA) - in p-globin chains in the sixth position, glutamic acid is replaced by valine. This is a consequence of the replacement of one of the bases in the triplet encoding glutamic acid (CTT or CTC). As a result, a triplet encrypting valine (CAT or CAC) appears. In this case, the replacement of one amino acid in the peptide significantly changes the properties of globin, which is part of hemoglobin (its ability to bind to 02 decreases), a person develops signs of sickle cell anemia.

In some cases, replacing one base with another can lead to the appearance of one of the nonsense triplets (ATT, ATC, ACT) that does not code for any amino acid. The consequence of such a replacement will be the interruption of the synthesis of the peptide chain. It is estimated that nucleotide substitutions in one triplet lead in 25% of cases to the formation of synonymous triplets; in 2-3 - meaningless triplets, in 70 - 75% - to the occurrence of true gene mutations.

Thus, base substitution mutations can arise both as a result of spontaneous changes in the base structure in one of the strands of an already existing DNA double helix, and during replication in a newly synthesized strand. If these changes are not corrected during reparation (or, conversely, occur during reparation), they are fixed in both chains and will then be reproduced in the next replication cycles. Therefore, an important source of such mutations are violations of the processes of replication and repair.

Mutations with a shift in the reading frame. This type of mutation makes up a significant proportion of spontaneous mutations. They occur due to the loss or insertion of one or more pairs of complementary nucleotides into the DNA nucleotide sequence. Most of the studied frameshift mutations were found in sequences consisting of identical nucleotides.

A change in the number of nucleotide pairs in a DNA chain is facilitated by the effects on the genetic material of certain chemicals, such as acridine compounds. By deforming the structure of the DNA double helix, they lead to the insertion of additional bases or their loss during replication. An example is the mutations obtained in the T4 phage when exposed to proflavin. They consist in the inclusion or removal of just one nucleotide pair. An important reason for the change in the number of nucleotide pairs in a gene according to the type of large divisions (fallouts) can be X-ray irradiation. In the fruit fly, for example, a mutation in the gene that controls the color of the eye is known, which is caused by irradiation and consists of a division of about 100 nucleotide pairs.

Fig.21. Pleiotropic effect of single amino acid substitution in the β-chain of human hemoglobin leading to the development of sickle cell anemia

A large number of insertion-type mutations occur due to the inclusion of mobile genetic elements, transposons, in the nucleotide sequence. Transposons are fairly long nucleotide sequences built into the genomes of eu- and prokaryotic cells that can spontaneously change their position. With a certain probability, insertions and divisions can occur as a result of recombination errors with unequal intragenic crossing over (Fig. 22).

Fig.22. Frameshift mutations (unequal exchange with intragenic crossing over):

I - breaks of allelpy genes in different areas and the exchange of fragments between them;

II - loss of the 3rd and 4th pairs of nucleotides, a shift in the reading frame;

III - doubling the 3rd and 4th pairs of nucleotides, shifting the reading frame

Fig.23. The consequence of a change in the number of nucleotide pairs in a DNA molecule

The shift of the reading frame as a result of the insertion of one nucleotide into the codogenic chain leads to a change in the composition of the peptide encrypted in it

With continuity of reading and non-overlapping of the genetic code, a change in the number of nucleotides, as a rule, leads to a shift in the reading frame and a change in the meaning of biological information recorded in a given DNA sequence (Fig. 23). However, if the number of nucleotides inserted or lost is a multiple of three, frameshift may not occur, but it will result in the inclusion of additional amino acids or the loss of some of them from the polypeptide chain. A possible consequence of the frameshift is the appearance of nonsense striplets, leading to the synthesis of shortened peptide chains.

Mutations according to the type of inversion of nucleotide sequences in the gene. This type of mutation occurs due to a 180° turn of a DNA segment. Usually, this is preceded by the formation of a loop by the DNA molecule, within which replication proceeds in the opposite direction to the correct one.

Within the inverted region, the reading of information is disturbed, as a result, the amino acid sequence of the protein changes.

4.2.4 Elementary units of variability genetic material. Mouton. Recon

A gene is an elementary unit of the function of hereditary material. This means that a fragment of a DNA molecule corresponding to an individual gene and determining, thanks to the biological information contained in it, the possibility of developing a particular trait, is further indivisible in a functional sense. The information about gene mutations outlined above indicates the significance of changes in the chemical structure that affect not the entire gene, but its individual sections, as a result of which new variants of the trait appear.

The minimum amount of hereditary material that, when changing, can lead to the appearance of variants of a trait, corresponds to the elementary unit of the mutation process and is called a muton. The examples of gene mutations discussed above indicate that it is enough to replace one pair of complementary bases in a gene in order to change the properties of the protein it encodes. Thus, a muton corresponds to one pair of complementary nucleotides.

Part of the gene mutations by the type of insertions and deletions of nucleotide pairs occurs due to the unequal exchange between DNA molecules during crossing over, i.e. in violation of recombination between them. This is accompanied by a shift in the reading frame and leads to a disruption in the synthesis of a peptide chain with desired properties. Observations show that the insertion or deletion of one pair of nucleotides is sufficient to distort the biological information recorded in the gene. From what has been said, it follows that the elementary unit of recombination, the recon, corresponds at the molecular level to one pair of nucleotides.

Changes in nucleotide sequences arising spontaneously or under the influence of various external influences lead to the fact that the same gene can exist in several variants that differ in the biological information contained in them. The specific form of existence of a gene, which determines the possibility of developing a specific variant of a given trait, is called an allele. The alleles of a gene are located in the same region - a locus - of a certain chromosome, which normally can simultaneously contain only one of a series of alleles. This makes alleles alternative (mutually exclusive) options for the existence of a gene.

Changes in the chemical structure can occur in different regions of the gene. If they are compatible with life, i.e. do not lead to the death of cells or organisms - carriers of these mutations, all of them are stored in the gene pool of the species.

The presence in the gene pool of a species at the same time different alleles of a gene is called multiple allelism. An example of this is the different eye color options in the fruit fly: white, cherry, red, apricot, eosin, due to different alleles of the corresponding gene. In humans, as in other representatives of the organic world, multiple allelism is characteristic of many genes. So, three alleles of the I gene determine the blood group according to the AB0 system (I A, I B, I 0). The gene that determines the Rh-belonging has two alleles. More than a hundred alleles have genes for α- and β-polypeptides of hemoglobin.

The cause of multiple allelism is random changes in the structure of the gene (mutations) that are preserved in the process of natural selection in the gene pool of the population. The diversity of alleles that recombine during sexual reproduction determines the degree of genotypic diversity among representatives of a given species, which is of great evolutionary importance, increasing the viability of populations under changing conditions of their existence. In addition to evolutionary and ecological significance, the allelic state of genes has a great influence on the functioning of the genetic material. In diploid somatic cells of eukaryotic organisms, most genes are represented by two alleles that together influence the formation of traits.

4.2.5 Functional classification of gene mutations

Changes in the structure of the gene, as a rule, are unfavorable, reducing the viability of the cell, organism (harmful mutations), and sometimes lead to their death (lethal mutations). Rarely occurring mutations do not significantly affect the viability of their carriers, so they are considered as neutral. Finally, alleles that have a beneficial effect (beneficial mutations) appear extremely rarely, providing their carriers with preferential survival. In most cases, the newly emerged allele of the gene acts as recessive in relation to the "wild" type allele common in nature, i.e. does not appear in combination with it. But sometimes the mutant form of a gene can be dominant, i.e. suppress the manifestation of the "wild" allele, which is more common in the gene pool of the population.

4.2.6 Mechanisms that reduce adverse effects gene mutations

As a result of gene mutations, the meaning of biological information changes. The consequences of this can be twofold. In environments that change little, new information usually reduces survival. With a sharp change in the conditions of existence, with the development of a new ecological niche, the availability of various information is useful. In this regard, the intensity of the mutation process under natural conditions is maintained at a level that does not cause a catastrophic decrease in the viability of the species. An important role in limiting the adverse effects of mutations belongs to anti-mutation mechanisms that have arisen in evolution.

Some of these mechanisms are discussed above. We are talking about the features of the functioning of DNA polymerase, which selects the required nucleotides in the process of DNA replication, and also performs self-correction during the formation of a new DNA strand, along with an editing endonuclease. Various mechanisms of DNA structure repair and the role of the degeneracy of the genetic code are analyzed in detail. The solution to this problem is the triplet nature of the biological code, which allows the minimum number of substitutions within the triplet, leading to information distortion. Thus, 64% of the substitutions of the third nucleotide in triplets do not change their semantic meaning. True, substitutions of the second nucleotide in 100% lead to a distortion of the meaning of the triplet.

The pairing of chromosomes in the diploid karyotype of eukaryotic somatic cells serves as a protection factor against the adverse consequences of gene mutations.

The pairing of alleles of genes prevents the phenotypic manifestation of mutations if they are recessive.

A certain contribution to the reduction of the harmful effects of gene mutations is made by the phenomenon of extracopying of genes encoding vital macromolecules. It consists in the presence in the genotype of several tens, and sometimes hundreds of identical copies of such genes. An example is the genes of rRNA, tRNA, histone proteins, without which the vital activity of any cell is impossible.

In the presence of extracopies, a mutational change in one or even several identical genes does not lead to catastrophic consequences for the cell. Copies that remain unchanged are enough to ensure normal functioning.

The functional inequivalence of amino acid substitutions in the polypeptide is also essential. If the new and replaced amino acids are similar in physicochemical properties, changes in the tertiary structure and biological properties of the protein are insignificant.

Thus, mutant human HbS and HbC hemoglobins differ from normal HbA hemoglobin by the replacement of the glutamic acid p-chain in the 6th position with valine or lysine, respectively. The first replacement dramatically changes the properties of hemoglobin and leads to the development of a serious illness - sickle cell anemia.

With the second replacement, the properties of hemoglobin change to a much lesser extent.

The reason for these differences is that glutamic acid and lysine exhibit similar hydrophilic properties, while valine is a hydrophobic amino acid.

Thus, these mechanisms contribute to the preservation of genes selected during evolution and, at the same time, the accumulation of their various alleles in the gene pool of a population, forming a reserve of hereditary variability. The latter determines the high evolutionary plasticity of the population, i.e. the ability to survive in a variety of conditions.

4.3 Use of genetic information in life processes

4.3.1 The role of RNA in the realization of hereditary information

Hereditary information, written down with the help of the genetic code, is stored in DNA molecules and multiplies in order to provide newly formed cells with the necessary "instructions" for their normal development and functioning. At the same time, DNA does not directly participate in the life support of cells. The role of an intermediary, whose function is to translate the hereditary information stored in DNA into a working form, is played by ribonucleic acids - RNA.

Unlike DNA molecules, ribonucleic acids are represented by one polynucleotide chain, which consists of four types of nucleotides containing sugar, ribose, phosphate and one of the four nitrogenous bases - adenine, guanine, uracil or cytosine. RNA is synthesized on DNA molecules using RNA polymerase enzymes in compliance with the principle of complementarity and antiparallelism, and uracil is complementary to DNA adenine in RNA. The whole variety of RNAs acting in the cell can be divided into three main types: mRNA, tRNA, rRNA.

Matrix, or information, RNA (mRNA, or mRNA). Transcription. In order to synthesize proteins with desired properties, an "instruction" about the order in which amino acids are included in the peptide chain comes to the site of their construction. This instruction is contained in the nucleotide sequence of matrix, or information RNA (mRNA, mRNA) synthesized at the corresponding DNA regions. The process of mRNA synthesis is called transcription.

The synthesis of mRNA begins with the discovery by RNA polymerase of a special site in the DNA molecule, which indicates the site of the start of transcription - the promoter. After attaching to the promoter, RNA polymerase unwinds the adjacent turn of the DNA helix. Two strands of DNA diverge at this point, and on one of them the enzyme synthesizes mRNA. The assembly of ribonucleotides into a chain occurs in compliance with their complementarity with DNA nucleotides, and also antiparallel to the template DNA chain. Due to the fact that RNA polymerase is able to assemble a polynucleotide only from the 5' end to the 3' end, only one of the two DNA strands can serve as a template for transcription, namely the one that faces the enzyme with its 3' end ( 3 "→ 5"). Such a chain is called codogenic (Fig. 3.24). The antiparallelism of the connection of two polynucleotide chains in a DNA molecule allows RNA polymerase to correctly select a template for mRNA synthesis.

Moving along the codogenic DNA chain, RNA polymerase carries out a gradual, precise rewriting of information until it encounters a specific nucleotide sequence - a transcription terminator. In this region, RNA polymerase is separated both from the DNA template and from the newly synthesized mRNA (Fig. 25). A fragment of a DNA molecule, including a promoter, a transcribed sequence and a terminator, forms a transcription unit - a transcripton.

During synthesis, as the RNA polymerase moves along the DNA molecule, the single-stranded sections of DNA it has passed through are again combined into a double helix. The mRNA formed during transcription contains an exact copy of the information recorded in the corresponding section of DNA. Three adjacent mRNA nucleotides that code for amino acids are called codons. The mRNA codon sequence codes for the sequence of amino acids in the peptide chain. The mRNA codons correspond to certain amino acids (Table 1).

Table 1. Genetic code of mRNA (terminator codons are underlined). Second nucleotide

At		C		BUT		G

Fig.24. mRNA synthesis scheme

The template for mRNA transcription is the codogenic DNA strand facing the enzyme with its 3-terminus

Rice. 25. The role of RNA polymerase in transcription:

I - detection of the promoter region in the DNA molecule and unwinding of the DNA helix; II - initiation of the synthesis of the RNA chain by binding the first two ribonucleoside gryphosphates; III - extension of the RNA chain in the direction 5 "→ 3" by attaching ribonucleoside gryphosphates; IV - release of the 5" end of the synthesized RNA and restoration of the DNA double helix; V - completion of RNA synthesis in the terminator region, separation of the polymerase from the completed RNA chain

Transfer RNA (tRNA). Broadcast. Transfer RNA (tRNA) plays an important role in the process of using hereditary information by the cell. Delivering the necessary amino acids to the assembly site of peptide chains, tRNA acts as a translational mediator.

tRNA molecules are polynucleotide chains synthesized on specific DNA sequences. They consist of a relatively small number of nucleotides - 75-95. As a result of the complementary connection of bases that are located in different parts of the tRNA polynucleotide chain, it acquires a structure resembling a clover leaf in shape (Fig. 26).

Fig.26. The structure of a typical tRNA molecule

It has four main parts that perform different functions. The acceptor "stalk" is formed by two complementary connected terminal parts of tRNA. It is seven base pairs. The middle of these branches - the anticodon - consists of five pairs of nucleotides and contains an anticodon in the center of its loop.The anticodon is three nucleotides complementary to the mRNA codon, which encodes the amino acid transported by this tRNA to the site of peptide synthesis.

Between the acceptor and anticodon branches are two side branches. In their loops, they contain modified bases - dihydrouridine (D-loop) and the TψC triplet, where y is pseudouriain (T^C-loop). Between the aiticodone and T^C branches there is an additional loop, which includes from 3-5 to 13-21 nucleotides.

In general, different types of tRNA are characterized by a certain constancy of the nucleotide sequence, which most often consists of 76 nucleotides. The variation in their number is mainly due to the change in the number of nucleotides in the additional loop. Complementary regions that support the tRNA structure are usually conserved. The primary structure of tRNA, determined by the sequence of nucleotides, forms the secondary structure of tRNA, which has the shape of a clover leaf. In turn, the secondary structure determines the three-dimensional tertiary structure, which is characterized by the formation of two perpendicular double helixes (Fig. 27). One of them is formed by the acceptor and TψC branches, the other by the anticodon and D branches.

At the end of one of the double helixes is the transported amino acid, at the end of the other is the anticodon. These areas are the most remote from each other. The stability of the tertiary structure of tRNA is maintained due to the appearance of additional hydrogen bonds between the bases of the polynucleotide chain, located in different parts of it, but spatially close in the tertiary structure.

Different types of tRNAs have a similar tertiary structure, although with some variations.

Fig.27. Spatial organization of tRNA:

I - the secondary structure of tRNA in the form of a "clover leaf", determined by its primary structure (the sequence of nucleotides in the chain);

II - two-dimensional projection of the tertiary structure of tRNA;

III - layout of the tRNA molecule in space

One of the features of tRNA is the presence in it of unusual bases that arise as a result of chemical modification after the inclusion of a normal base in the polynucleotide chain. These altered bases determine the great structural diversity of tRNAs in the general plan of their structure. Of greatest interest are modifications of the bases that form the anticodon, which affect the specificity of its interaction with the codon. For example, the atypical base inosine, sometimes in the 1st position of the tRNA anticodon, is able to complementarily combine with three different third bases of the mRNA codon - U, C and A (Fig. 3.28). Since one of the features of the genetic code is its degeneracy (see section 3.4.1.2), many amino acids are encoded by several codons, which, as a rule, differ in their third base. Due to the nonspecific binding of the modified anticodon base, one tRNA recognizes several synonymous codons.

Fig.28. Hydrogen bonding of inosine to three different nitrogenous bases Hydrogen bonds are indicated by dots

The existence of several types of tRNAs capable of binding to the same codon has also been established. As a result, not 61 (by the number of codons), but about 40 different tRNA molecules are found in the cytoplasm of cells. This amount is enough to transport 20 different amino acids to the protein assembly site.

Along with the function of precise recognition of a certain codon in mRNA, the tRNA molecule delivers a strictly defined amino acid encrypted with this codon to the site of synthesis of the peptide chain. The specific connection of tRNA with "its" amino acid proceeds in two stages and leads to the formation of a compound called aminoacyl-tRNA (Fig. 29).

Fig.29. Attachment of an amino acid to the corresponding tRNA:

I - 1st stage, the interaction of amino acids and ATP with the release of pyrophosphate;

II - 2nd stage, attachment of the adipated amino acid to the 3" end of the RNA

At the first stage, the amino acid is activated by interacting with its carboxyl group with ATP. As a result, an adipylated amino acid is formed.

At the second stage, this compound interacts with the OH group located at the 3 "end of the corresponding tRNA, and the amino acid attaches its carboxyl group to it, releasing AMP. Thus, this process proceeds with the expenditure of energy obtained during the hydrolysis of ATP to AMP .

The specificity of the combination of an amino acid and a tRNA carrying the corresponding anticodon is achieved due to the properties of the enzyme aminoacyl-tRNA synthetase. In the cytoplasm, there is a whole set of such enzymes that are capable of spatial recognition, on the one hand, of their amino acid, and on the other hand, of the corresponding tRNA anticodon (Fig. 3.30). Hereditary information, "recorded" in DNA molecules and "rewritten" in mRNA, is deciphered during translation due to two processes of specific recognition of molecular surfaces. First, the enzyme aminoacyl-tRNA synthetase ensures the connection of tRNA with the amino acid it transports. The aminoacyl-tRNA then pairs complementarily with the mRNA through anticodon-codon interaction. With the help of the tRNA system, the language of the mRNA nucleotide chain. translated into the language of the amino acid sequence of the peptide (Fig. 30).

Ribosomal RNA (rRNA). Ribosomal cycle of protein synthesis. The process of interaction between mRNA and tRNA, which ensures the translation of information from the language of nucleotides into the language of amino acids, is carried out on ribosomes. The latter are complex complexes of rRNA and various proteins, in which the former form a scaffold. Ribosomal RNAs are not only a structural component of ribosomes, but also ensure their binding to a specific mRNA nucleotide sequence. This sets the start and reading frame for the formation of the peptide chain. In addition, they provide interaction between the ribosome and tRNA. Numerous proteins that make up ribosomes, along with rRNA, perform both structural and enzymatic roles.

Fig.30. Scheme of translation of the genetic code: I - attachment of an amino acid (tryptophan) to the corresponding tRNA using the enzyme aminoacyl-tRNA synthetase; II - attachment of tRNA carrying its amino acid to mRNA due to the binding of its anticodon to the mRNA codon

The ribosomes of pro- and eukaryotes are very similar in structure and function. They consist of two subparticles: large and small. In eukaryotes, the small subunit is formed by one rRNA molecule and 33 different protein molecules. The large subunit combines three rRNA molecules and about 40 proteins. Prokaryotic ribosomes and mitochondrial and plastid ribosomes contain fewer components.

Ribosomes have two grooves. One of them holds the growing polypeptide chain, the other - mRNA. In addition, two tRNA-binding sites are isolated in ribosomes. Aminoacyl-tRNA is located in the aminoacyl, A-site, carrying a specific amino acid. In the peptidyl, P-section, tRNA is usually located, which is loaded with a chain of amino acids connected by peptide bonds. The formation of A- and P-sites is provided by both subunits of the ribosome.

At each moment, the ribosome shields a segment of mRNA with a length of about 30 nucleotides. This ensures the interaction of only two tRNAs with two adjacent mRNA codons (Fig. 31).

The translation of information into the "language" of amino acids is expressed in the gradual build-up of the peptide chain in accordance with the instructions contained in the mRNA. This process takes place on ribosomes, which provide the sequence for deciphering information using tRNA. Three phases can be distinguished during translation: initiation, elongation, and termination of peptide chain synthesis.

Fig.31. Binding sites of tRNA molecules and ribosome:

I - unloaded ribosome, II - loaded ribosome; ak - amino acid

The initiation phase, or the beginning of peptide synthesis, consists in combining two ribosome subparticles that were previously separated in the cytoplasm at a certain mRNA site and attaching the first aminoacyl-tRNA to it. This also sets the frame for reading information contained in mRNA (Fig. 32).

In the molecule of any mRNA, near its 5 "end, there is a site that is complementary to the rRNA of the small subunit of the ribosome and specifically recognized by it. Next to it is the initiating start codon AUT, which encodes the amino acid methionine. The small subunit of the ribosome connects to the mRNA in such a way that the start codon AUT is located in the region corresponding to the P-site.At the same time, only the initiating tRNA carrying methionine is able to take a place in the unfinished P-section of the small subunit and complementary connect to the start codon.After the described event, the large and small subunits of the ribosome combine to form its peptidyl and aminoacyl plots (Fig. 3.32).

Fig.32. Initiation of protein synthesis:

I - connection of a small subchapshchy of the ribosome with mRNA, attachment to the start codon of the tRNA carrying methionine, which is located in the unfinished P-section; II - connection of large and small subparticles of the ribosome with the formation of P - and A-sites; the next stage is associated with the placement in the A-site of the aminoacyl-tRNA corresponding to the mRNA codon located in it, the beginning of elongation; ak - amino acid

By the end of the initiation phase, the P-site is occupied by aminoacyl-tRNA associated with methionine, while the A-site of the ribosome is located next to the start codon.

The described processes of translation initiation are catalyzed by special proteins - initiation factors, which are movably associated with a small subunit of the ribosome. Upon completion of the initiation phase and the formation of the ribosome-mRNA-initiating aminoacyl-tRNA complex, these factors are separated from the ribosome.

The elongation phase, or peptide elongation, includes all reactions from the formation of the first peptide bond to the attachment of the last amino acid. It is a cyclically recurring event in which there is a specific recognition of the next codon aminoacyl-tRNA located in the A-site, a complementary interaction between the anticodon and codon.

Due to the peculiarities of the three-dimensional organization of tRNA when its anticodon is connected to the mRNA codon. the amino acid transported by it is located in the A-site, in the vicinity of the previously included amino acid located in the P-site. A peptide bond is formed between two amino acids, catalyzed by special proteins that make up the ribosome. As a result, the previous amino acid loses its connection with its tRNA and joins the aminoacyl-tRNA located in the A-site. The tRNA located at this moment in the P-site is released and goes into the cytoplasm (Fig. 33). The movement of tRNA loaded with a peptide chain from the A site to the P site is accompanied by the advancement of the ribosome along the mRNA by a step corresponding to one codon. Now the next codon comes into contact with the A site, where it will be specifically "recognized" by the corresponding aminoacyl-tRNA, which will place its amino acid there. This sequence of events is repeated until the A-site of the ribosome receives a terminating codon for which no corresponding tRNA exists.

Fig.33. The elongation phase in protein synthesis:

1st stage - aminoacyl-tRNA joins the codon located in the A-site;

2nd stage - between the amino acids located in the A- and P-sites, a peptidial bond is formed: the tRNA located in the P-site is released from its amino acid and leaves the ribosome;

3rd stage - the ribosome moves along the mRNA by one codon so that the tRNA loaded with the peptide chain moves from the A-site to the P-site; free A-site can be occupied by the corresponding aminoacyl-tRNA

Fig.34. Termination of peptide chain synthesis:

1st stage - attachment of the release factor to the stop codon;

2nd stage - termination, release of the completed peptide;

3rd stage - dissociation of the ribosome into two subparticles

The assembly of the peptide chain is carried out at a fairly high rate, depending on temperature. In bacteria at 37 °C, it is expressed as the addition of 12 to 17 amino acids per 1 s to the subdipeptide. In eukaryotic cells, this rate is lower and is expressed as the addition of two amino acids in 1 s.

The termination phase, or the completion of polypeptide synthesis, is associated with the recognition by a specific ribosomal protein of one of the termination codons (UAA, UAG, or UGA) when it enters the A-site zone of the ribosome. In this case, water is attached to the last amino acid in the peptide chain, and its carboxyl end is separated from the tRNA. As a result, the completed peptide chain loses its connection with the ribosome, which splits into two subparticles (Fig. 34).

4.3.2 Features of organization and expression genetic information in pro- and eukaryotes

According to the chemical organization of the material of heredity and variability, eukaryotic and prokaryotic cells do not fundamentally differ from each other. Their genetic material is represented by DNA. Common to them is the principle of recording genetic information, as well as the genetic code. The same amino acids are encrypted in pro- and eukaryotes with the same codons. In principle, the use of hereditary information stored in DNA is carried out in the same way in these types of cells. First, it is transcribed into the nucleotide sequence of the mRNA molecule, and then translated into the amino acid sequence of the peptide on ribosomes with the participation of tRNA. However, some features of the organization of hereditary material, which distinguish eukaryotic cells from prokaryotic ones, cause differences in the use of their genetic information.

The hereditary material of a prokaryotic cell is contained mainly in a single circular DNA molecule. It is located directly in the cytoplasm of the cell, where there are also tRNAs and enzymes necessary for gene expression, some of which are contained in ribosomes. Prokaryotic genes consist entirely of coding nucleotide sequences that are realized during the synthesis of proteins, tRNA or rRNA.

The hereditary material of eukaryotes is larger in volume than that of prokaryotes. It is located mainly in special nuclear structures - chromosomes, which are separated from the cytoplasm by the nuclear envelope. The apparatus necessary for protein synthesis, consisting of ribosomes, tRNA, a set of amino acids and enzymes, is located in the cytoplasm of the cell.

Significant differences exist in the molecular organization of genes in eukaryotic cells. In most of them, exon coding sequences are interrupted by intron regions that are not used in the synthesis of tRNA, rRNA, or peptides. The number of such regions varies in different genes. It has been established that the chicken ovalbumin gene includes 7 introns, and the mammalian procollagen gene - 50. These regions are removed from the primary transcribed RNA, and therefore the use of genetic information in a eukaryotic cell occurs somewhat differently. In a prokaryotic cell, where the hereditary material and the apparatus for protein biosynthesis are not spatially separated, transcription and translation occur almost simultaneously. In a eukaryotic cell, these two stages are not only spatially separated by the nuclear envelope, but they are also separated in time by the processes of mRNA maturation, from which uninformative sequences must be removed (Fig. 35).

Rice. 35. Generalized scheme of the process of expression of genetic information in a eukaryotic cell

In addition to these differences at each stage of the expression of genetic information, some features of the course of these processes in pro- and eukaryotes can be noted.

Transcription in pro- and eukaryotes. Transcription is the synthesis of RNA on a DNA template. In prokaryotes, the synthesis of all three types of RNA is catalyzed by one complex protein complex - RNA polymerase.

The transcriptional apparatus of eukaryotic cells includes three nuclear RNA polymerases, as well as mitochondrial and plastid RNA polymerases. RNA polymerase I is found in the nucleoli of cells and is responsible for the transcription of rRNA genes. RNA polymerase II is located in the nuclear sap and is responsible for the synthesis of the mRNA precursor. RNA polymerase III is a small fraction found in the nuclear sap and is involved in the synthesis of small rRNAs and tRNAs. Each of these enzymes has two large subunits and up to 10 small ones. The RNA polymerases of mitochondria and plastids differ from nuclear ones.

The enzyme complex of RNA polymerase specifically recognizes a certain nucleotide sequence (often more than one) located at a certain distance from the starting point of transcription - the promoter. The starting point is the DNA nucleotide that corresponds to the first nucleotide included by the enzyme in the RNA transcript.

In prokaryotes, usually not far from the starting point against the course of transcription is a sequence of six nucleotides - TATAAT, called the Pribnow block. This is an average sequence consisting of the most frequently occurring bases, the most conservative of which are the 1st, 2nd and 6th bases. The presence in this sequence of bases, predominantly connected by double hydrogen bonds with complementary bases of another strand, obviously facilitates the local melting of the DNA double helix and the formation of its two single-stranded regions upon contact with RNA polymerase. The Pribnov block is located in the position from - 11 to - 5 or from - 14 to - 8, i.e. a few nucleotides before the starting point of transcription (Fig. 36). Upon detecting this sequence, RNA polymerase binds strongly to it and starts RNA synthesis. An equally important role in establishing contact between RNA polymerase and DNA belongs to another nucleotide sequence, the center of which is in position - 35. It is called the recognition region - TTGACA. Between the two indicated areas, the distance is quite constant and ranges from 16 to 19 base pairs (bp).

Eukaryotic gene promoters also include at least two specific nucleotide sequences centered at -25 bp and -75 bp.

At a distance of 19-27 nucleotides from the starting point against the course of transcription in many eukaryotic genes, the average statistical sequence TAT A T A A T (TATA block, or Hogness block) was found, in which, as in the Pribnow block in prokaryotes, bases predominate, forming weaker bonds. The second sequence, found in many eukaryotic promoters and consisting of GG C T CAATCT, is referred to as the CAAT block. It occupies a position between -70 and -80 nucleotides and is also the region recognized by the polymerase. In some genes, multicomponent promoters have been found.

Thus, in individual genes of the herpes virus, three DNA sequences are required for effective transcription initiation, located between -19 and -27, between -47 and -61, and also between -80 and -105 nucleotides.

Fig.36. Contact points for RNA polymerase located in the upper strand of DNA (promoter)

Features of the promoter regions indicate that not only the combination of bases in certain regions of the promoter is important for transcription initiation, but also the relative position in the DNA molecule of these regions, to which the RNA polymerase enzyme complex binds.

After establishing contact between the RNA polymerase and the promoter site, the assembly of the RNA molecule begins, which most often includes the first nucleotide carrying a purine base (usually adenine) and containing three 5'-phosphate residues.

Further, as the RNA polymerase moves along the DNA molecule, a gradual elongation of the RNA chain occurs, which continues until the enzyme encounters the terminator region. The terminator is the site where the further growth of the RNA chain stops and it is released from the DNA template. The RNA polymerase is also separated from the DNA, which rebuilds its double-stranded structure.

Fig.37. A region of DNA with double symmetry - a palindrome:

I - palindrome, in which there is a sequence that is the same when reading in opposite directions;

II - a palindrome in which the shaded inverted repeat is at a distance from the axis of symmetry

In prokaryotic cells, terminators necessarily contain palindromes - double-stranded sequences of DNA nucleotides that read equally in both directions (Fig. 37). An RNA region transcribed from such a sequence is able to form double-stranded hairpins due to the complementary pairing of palindrome nucleotides. Perhaps this is the signal for the completion of transcription, recognized by RNA polymerase (Fig. 3.38). The resulting hairpins apparently stop the polymerase at the terminator. Following the hairpin, the RNA molecule includes a sequence of nucleotides containing uracil (polyU), which probably takes part in the release of RNA from the DNA template. Indeed, a polyU RNA sequence linked to a polyadenyl (polyA) DNA sequence is characterized by a weak interaction. It is noteworthy that the DNA region rich in A-T pairs is found not only at the site of transcription initiation (Pribnov block), but also in the terminator region.

Bacterial terminators vary considerably in their effectiveness. Some of them, as it were, are not noticed by RNA polymerase, and it continues transcription outside the terminator. Such reading of the terminator during transcription of bacterial genes is observed as a result of the prevention of termination by specific proteins - anti-termination factors. The consequence of anti-termination is the synthesis of polycistronic mRNA, which includes information written off from several successively located structural genes.

Terminators of eukaryotic genes have been studied to a lesser extent than in proskaryotes, but they also contain regions rich in G-C pairs connected by triple hydrogen bonds, in which a site with A-T pairs is located. At this site, the transcript includes a polyU sequence that weakly interacts with the template polyA region of DNA.

It is possible that the terminator region, rich in G-C pairs, plays a certain role in stopping RNA polymerase, and the RNA region containing UUUU ensures the separation of the transcript from the DNA template.

In eukaryotes, the formation of structures similar to hairpins in prokaryotic RNAs was not found. Therefore, how transcription termination occurs in them remains unclear.

All mRNAs contain coding regions representing a set of codons that encode the amino acid sequence in the peptide. As a rule, these regions begin with the start codon AUG, but sometimes the GUT codon is used in bacteria. At the end of the coding sequence is a termination codon. In addition to the coding regions in mRNA, additional sequences can be located at both ends. At the 5" end, this is the leader region, located before the start codon. At the 3" end, there is a trailer following the terminator codon.

Fig.38. Hairpin formation by an RNA region during transcription termination in prokaryotes

The RNA region carrying the palindrome forms a complementary pairing structure - a hairpin (inverted repeats are shaded)

In the polycistronic mRNA of prokaryotes, there are intercistronic regions between the coding regions, which vary in size (Fig. 3.39).

Fig.39. Polycistronic messenger RNA of prokaryotes:

1 - non-coding regions, 2 - intercistronic regions, 3 - coding regions, 4 - termination codons

Due to the fact that prokaryotic genes consist entirely of nucleotide sequences involved in encoding information, the RNA transcribed from them immediately after their synthesis is able to function as templates for translation. Only in exceptional cases is their preliminary maturation required - processing.

Unlike prokaryotic genes, most of the genes of eukaryotic cells are discontinuous, since they carry in their composition non-informative nucleotide sequences - introns that are not involved in encoding information. In this regard, the primary transcripts synthesized by RNA polymerase II are larger than necessary for translation and are less stable. Together, they form the so-called heterogeneous nuclear RNA (tRNA), which, before leaving the nucleus and beginning to function actively in the cytoplasm, undergoes processing and turns into mature mRNA.

eukaryotic mRNA processing. The maturation, or processing, of mRNA involves the modification of the primary transcript and the removal of non-coding intron regions from it, followed by the connection (splicing) of coding sequences - exons. Modification of the primary transcript of eukaryotic mRNA begins shortly after the synthesis of its 5 "end containing one of the purine bases (adenine or guanine). A cap is formed at this end, which blocks the 5" end of the mRNA by attaching to the first nucleotide of the triphosphonucleoside transcript containing guanine, bond 5 "-5".

Gfff + fffAfN… → GfffAfN. + ff + f As a result, the GfffAfFM... sequence is formed, in which the tuanine residue is in the reverse orientation with respect to other mRNA nucleotides. Modification of the 5 "end of mRNA also involves methylation of the attached guanine and the first two or three bases of the primary transcript (Fig. 3.40). The caps formed at the 5" ends of the mRNA ensure the recognition of mRNA molecules by small subparticles of ribosomes in the cytoplasm. Caching is carried out even before the end of the synthesis of the primary transcript.

Rice. 40. Formation of mature eukaryotic mRNA during processing:

1 - non-coding sequences, 2 - exons, 3 - introns, 4 - terminator codon

After transcription is completed, a part of the nucleotides at the 3 "end of the primary transcript is removed and a sequence consisting of 100-200 adenylic acid (polyA) residues (Fig. 3.40) is attached to it. It is believed that this sequence contributes to further processing and transport of mature mRNA from nucleus After the release of mRNA into the cytoplasm, its polyA sequence is gradually shortened under the action of enzymes that cleave nucleotides at the 3' end. Thus, the length of the polyA sequence can indirectly judge the residence time of mRNA in the cytoplasm. Possibly, the addition of the polyA sequence during processing enhances mRNA stability. However, about a third of mRNAs do not contain a polyA site at all. These include, for example, histone mRNAs.

The formation of a cap at the 5' end and a polyA sequence at the 3' end is characteristic only for the processing of RNA synthesized by RNA polymerase II. In addition to methylation during the formation of caps in mRNA of higher eukaryotes, methylation of a small part of internal nucleotides occurs at a frequency of approximately one per thousand bases of mRNA.

Along with the modification of eukaryotic mRNA, processing involves the removal from primary transcripts of intron regions that are not informative for a given protein, the size of which varies from 100 to 10,000 nucleotides or more. Introns account for about 80% of all hnRNA. The removal of introns followed by the joining of exonic regions is called splicing (Fig. 40).

Splicing is a mechanism that must ensure the removal of well-defined intron regions from the primary transcript. Violation of this process can lead to a shift in the reading frame during translation and the impossibility of synthesizing a normal peptide. The regularity of excision of introns is apparently ensured by the presence of specific nucleotide sequences at their ends, which serve as signals for splicing.

Several plausible splicing mechanisms have now been described to ensure the accuracy of this process. Perhaps it is achieved by the action of some enzymes that specifically recognize the terminal sections of introns and catalyze the breaking of phosphodiester bonds at the exon-intron border, and then the formation of bonds between two exons.

An active participation in splicing of special small, nuclear RNAs (snRNAs) that form complexes with proteins (snRNPs) has been established. Obviously, snRNAs interact complementarily with their nucleotide sequences with the terminal regions of introns, which form closed loops. Cleavage of RNA at the mouth of the intron loop leads to the removal of an uninformative sequence and the joining (splicing) of the adjacent exon ends.

The autocatalytic ability of the RNA transcript for splicing is also discussed. The described methods of splicing indicate the absence of a universal mechanism for this process, however, in all cases, accurate removal of introns is achieved with the formation of a specific mRNA that provides the synthesis of the protein necessary for the cell.

At present, the possibility of alternative (mutually exclusive) splicing has been proven, in which different nucleotide sequences can be deleted from the same primary transcript and different mature mRNAs can be formed. As a result, the same DNA nucleotide sequence can serve as information for the synthesis of different peptides. Alternative splicing is probably very typical in the mammalian immunoglobulin gene system, where it allows the formation of mRNA based on a single transcript for the synthesis of different types of antibodies.

Due to the transformations that occur with the RNA transcript during processing, mature eukaryotic mRNAs are more stable than prokaryotic mRNAs.

Upon completion of processing, mature mRNA is selected before entering the cytoplasm, where only 5% of hnRNA enters. The rest is split without leaving the nucleus.

Thus, transformations of the primary transcripts of eukaryotic genes, due to their exonitronic organization and the need for mRNA transfer from the nucleus to the cytoplasm, determine the features of the implementation of genetic information in a eukaryotic cell.

Translation in pro- and eukaryotes. In prokaryotic cells, the process of translation is associated with the synthesis of mRNA: they occur almost simultaneously. To a large extent, this is due to the fragility of bacterial mRNA, which is rapidly degraded. The relationship between transcription and translation in bacteria is manifested in the consistency of the rates of these processes. At 37°C, transcription occurs at a rate of 2500 nucleotides/min (14 codons/s), and translation occurs at a rate of 15 amino acids/s.

Translation in prokaryotes begins shortly after the formation of the 5 "end of mRNA, before its synthesis ends. As a result, following the RNA polymerase, ribosomes move along the mRNA, assembling peptide chains (Fig. 41). Some time after the start of transcription (about 1 min) and before the completion of translation of the 3'-end of the template, degradation of its 5'-end begins. Due to the fact that the lifetime of different mRNAs is not the same, the amount of protein synthesized on different templates is different.

One of the features of translation in prokaryotes is the inclusion in the peptide chain as the first amino acid of a modified methionine - formylmethionine, from which all newly synthesized peptides begin. Even in the case when the role of the start codon is performed by the code GUG, which encodes valine under normal conditions, formylmethionine appears in the first position of the peptide. The start codon AUG or GUG follows the leader site, which is shielded by the ribosome at the time of translation initiation.

The connection of the ribosome with mRNA is due to the complementary interaction of the nucleotides of one of the rRNAs with the nucleotide sequence of the mRNA leader.

This sequence (Shine-Dalgarno) is located 4-7 bases before the AUG codon and is found ubiquitously in leader regions in prokaryotes.

When the 5'-end of the mRNA is connected to the small subunit of the ribosome, the start codon usually appears almost in the middle of the mRNA fragment shielded by the ribosome, in the region corresponding to its P-site.

In eukaryotes, translation takes place in the cytoplasm, where mature mRNA enters from the nucleus. The copied end of the mRNA is recognized by the small subunit of the ribosome, then the leading sequence, containing up to 100 nucleotides, interacts with the rRNA. In this case, the start codon AUG is in the unfinished P-site of the ribosome. After aminoacyl-tRNA carrying methionine is attached to the start codon, two subunits of the ribosome are reunited and its A- and P-sites are formed. Protein synthesis in a eukaryotic cell, carried out on monocistronic mRNA, is completed after the passage of the ribosome through the entire mRNA, until it recognizes the terminator codon that stops the formation of peptide bonds.

Post-translational transformations of proteins. Peptide chains synthesized during translation, on the basis of their primary structure, acquire a secondary and tertiary, and many also a quaternary organization formed by several peptide chains. Depending on the functions performed by proteins, their amino acid sequences can undergo various transformations, forming functionally active protein molecules.

Many membrane proteins are synthesized as preproteins with a leader sequence at the N-terminus that provides him with membrane recognition. This sequence is cleaved off during maturation and incorporation of the protein into the membrane. Secretory proteins also have a leader sequence at the N-terminus that ensures their transport across the membrane. Some proteins, immediately after translation, carry additional amino acid prosequences that determine the stability of active protein precursors. During protein maturation, they are removed, allowing the transition of the inactive proprotein to the active protein. For example, insulin is initially synthesized as preproinsulin. During secretion, the pre-sequence is cleaved off, and then proinsulin undergoes a modification in which part of the chain is removed from it and it turns into mature insulin.

Fig.41. Transcription, translation and degradation of mRNA in prokaryotes:

I - RNA polymerase binds to DNA and begins to synthesize mRNA in the direction 5 "→ 3";

II - as the RNA polymerase advances, ribosomes are attached to the 5' end of the mRNA, starting protein synthesis;

III - a group of ribosomes follows the RNA polymerase, its degradation begins at the 5' end of the mRNA;

IV - the degradation process is slower than transcription and translation;

V - after the end of transcription, mRNA is released from DNA, translation and degradation at the 5 "end continue on it

Forming a tertiary and quaternary organization in the course of post-translational transformations, proteins acquire the ability to actively function, being included in certain cellular structures and performing enzymatic and other functions.

The considered features of the implementation of genetic information in pro- and eukaryotic cells reveal the fundamental similarity of these processes. Consequently, the mechanism of gene expression associated with transcription and subsequent translation of information that is encrypted with the help of a biological code has developed as a whole even before these two types of cellular organization were formed. The divergent evolution of the genomes of pro- and eukaryotes led to differences in the organization of their hereditary material, which could not but affect the mechanisms of its expression.

The constant improvement of our knowledge about the organization and functioning of the material of heredity and variability determines the evolution of ideas about the gene as a functional unit of this material.

Block 2. DNA. Questions 5,6,7.

Structure of DNA. Model of J. Watson and F. Crick. Properties and functions of hereditary material.

Self-reproduction of genetic material. DNA replication.

Organization of hereditary material in pro- and eukaryotes. Classification of nucleotide sequences in the eukaryotic genome (unique, moderately repetitive, highly repetitive).

In 1868, the Swiss chemist F. Miescher discovered in cell nuclei isolated from pus, and later from salmon sperm, a substance that he called "nuclein" (from Latin nucleus - nucleus). Subsequently, R. Altmann (1889) reported that the "nuclein" isolated by F. Miescher consists of two fractions - protein and nucleic acids. Nucleic acids, like proteins, have a primary structure (by which is meant their nucleotide sequence) and a three-dimensional structure. Interest in the structure of DNA intensified when, at the beginning of the 20th century. there was an assumption that DNA, possibly genetic material. In 1952, Chargaff discovered the rule of complementarity, which was later named after the creator. It lies in the fact that:

1. The amount of adenine is equal to the amount of thymine, and guanine is equal to cytosine: A=T, G=C.
2. The number of purines is equal to the number of pyrimidines: A + G = T + C.
3. The number of bases with amino groups in position 6 is equal to the number of bases with keto groups in position 6: A+C=G+T.

Subsequently, an x-ray of the DNA was obtained by Wilkinson. And a little later, Watson and Crick in 1953 proposed their own model of DNA, for which, together with Wilkinson, they were awarded the Nobel Prize in 1962.

Basic principles of DNA structure.

1. A DNA nucleotide monomer consisting of a nitrogenous base, deoxyribose, and a phosphoric acid residue. Nitrogenous bases can be purine A, G or pyrimidine C, T.

2. Nitrogenous bases are attached to C1 carbon atom in the pentose molecule, and phosphate is attached to C5. The third atom always has a group HE.

3. When the phosphate of one nucleotide interacts with the deoxyribose hydroxyl of the other, phosphodiester bond.

4. The connection of nucleotides occurs through the OH of the pentose to the C3 position and the phosphate of the subsequent nucleotide.

5. DNA is a double polynucleotide chain. Two polynucleotide chains are linked by hydrogen bonds the principle of complimentary A-T and G-C. There are two hydrogen bonds between A and T, and three hydrogen bonds between T and C.

6. Antiparallelism. The 5 end of one chain is connected to the 3 end of the other chain.

7. The diameter of the DNA helix is ​​2 nm, and the pitch length is 3.4 nm. There are 10 base pairs per turn.

8. Primary Structure- polynucleotide chain.

secondary structure- two complementary antiparallel polynucleotide chains.

Tertiary structure- three-dimensional spiral.

9. DNA has the ability to replicate.

REPLICATION.

1 - DNA template chains; 2 - enzyme helicase, separating the chains of matrix DNA; 3 - DSB-proteins that prevent the reunion of DNA chains; 4 - primase; 5 - RNA primer (synthesized by RNA polymerase - primase); 6 - DNA polymerase synthesizing daughter chains; 7 - leading daughter strand of DNA; 8 - ligase connecting the Okazaki fragments of the lagging DNA strand; 9 - Okazaki fragment (150-200 nucleotides); 10 - topoisomerase

The synthesis of a new DNA molecule is carried out in a semi-conservative way. This means that the daughter molecule will contain one parent and one newly synthesized strand. Since DNA synthesis occurs on a single-stranded template, it is preceded by an obligatory temporary separation of the two strands, with the formation of a replication fork. Using an electron microscope, the region of replication was found to have the appearance of an eye inside the unreplicated DNA (a replication eye consisting of approximately 300 nucleotides).

Replicon- DNA fragment from the point of origin of replication to the point of its termination.

To unwind the DNA helix, special enzymes (proteins). Several enzymes take part in replication, each of which performs its own function.

DNA helicases (helicases) break hydrogen bonds between bases, split strands, and advance the replication fork.

Destabilizing proteins hold chains.

DNA is a topoisomerase. Recall that DNA is a helix. Accordingly, in order for the fork to move forward, the spiral must quickly unwind. But this will require a large loss of energy. In fact, this still does not happen. This is facilitated by DNA topoisomerases. They introduce single- and double-strand breaks into the chain, allowing the chains to separate, and then eliminate these breaks. Thanks to one of the DNA strands begins to revolve around the second strand. They are also involved in the uncoupling of rings formed during the replication of circular DNA.

Synthesis of DNA strands occurs with the help of DNA polymerase. But this enzyme has a peculiarity. It is able to add nucleotides to the 3 end of an existing chain. Such a pre-formed chain is called seed, which synthesizes primase. The RNA primer is different from the rest of the DNA strand because it contains ribose. The seed size is small. The seed that has performed its function is removed by a special enzyme, and the gap formed in this case is eliminated. DNA polymerase(in this case, instead of a seed, it uses the 3OH end of the adjacent DNA fragment).

DNA replication assumes that the synthesis of two strands occurs simultaneously. But in reality, things don't quite work out that way. Remember that chains are antiparallel. And the synthesis of a new chain can occur only in the direction from end 5 to end 3. Therefore, continuous synthesis occurs only on one chain (leading). On the second (lagging behind) it occurs in Okazaki fragments. The synthesis of each of the fragments is carried out using an RNA primer. The primers are then removed, the gaps are filled with DNA polymerase, and the fragments are crosslinked with an enzyme. ligase .

Structural and functional organization of DNA in pro- and eukaryotes

Study the tables, copy them into a workbook.

Nucleic acids are macromolecular substances consisting of mononucleotides, which are connected to each other in a polymer chain using 3",5" - phosphodiester bonds and packed in cells in a certain way.

Nucleic acids are biopolymers of two varieties: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Each biopolymer consists of nucleotides that differ in carbohydrate residue (ribose, deoxyribose) and one of the nitrogenous bases (uracil, thymine). Accordingly, nucleic acids got their name.

Structure of deoxyribonucleic acid

Nucleic acids have primary, secondary and tertiary structures.

Primary structure of DNA

The primary structure of DNA is a linear polynucleotide chain in which mononucleotides are connected by 3", 5" phosphodiester bonds. The starting material for assembling a nucleic acid chain in a cell is the nucleoside 5'-triphosphate, which, as a result of the removal of β and γ residues of phosphoric acid, is able to attach the 3'-carbon atom of another nucleoside. Thus, the 3" carbon atom of one deoxyribose covalently binds to the 5" carbon atom of another deoxyribose via one phosphoric acid residue and forms a linear polynucleotide chain of nucleic acid. Hence the name: 3", 5"-phosphodiester bonds. Nitrogenous bases do not take part in the connection of nucleotides of one chain (Fig. 1.).

Such a connection, between the phosphoric acid residue of one nucleotide and the carbohydrate of the other, leads to the formation of a pentose-phosphate backbone of the polynucleotide molecule, on which nitrogenous bases are added one after the other from the side. Their sequence in the chains of nucleic acid molecules is strictly specific for cells of different organisms, i.e. has a specific character (Chargaff's rule).

A linear DNA chain, the length of which depends on the number of nucleotides included in the chain, has two ends: one is called the 3 "end and contains a free hydroxyl, and the other, the 5" end, contains a phosphoric acid residue. The circuit is polar and can be 5"->3" and 3"->5". An exception is circular DNA.

The genetic "text" of DNA is made up of code "words" - triplets of nucleotides called codons. DNA segments containing information about the primary structure of all types of RNA are called structural genes.

Polynucleoditic DNA chains reach gigantic sizes, so they are packed in a certain way in the cell.

Studying the composition of DNA, Chargaff (1949) established important regularities concerning the content of individual DNA bases. They helped uncover the secondary structure of DNA. These patterns are called Chargaff's rules. Chargaff rules the sum of purine nucleotides is equal to the sum of pyrimidine nucleotides, i.e. A + G / C + T \u003d 1 the content of adenine is equal to the content of thymine (A = T, or A / T = 1); the content of guanine is equal to the content of cytosine (G = C, or G/C = 1); the number of 6-amino groups is equal to the number of 6-keto groups of bases contained in DNA: G + T = A + C; only the sum of A + T and G + C is variable. If A + T > G-C, then this is the AT-type of DNA; if G + C > A + T, then this is the GC type of DNA. These rules say that when building DNA, a rather strict correspondence (pairing) must be observed not for purine and pyrimidine bases in general, but specifically for thymine with adenine and cytosine with guanine. Based on these rules, among other things, in 1953 Watson and Crick proposed a model of the secondary structure of DNA, called the double helix (Fig.).

Secondary structure of DNA

The secondary structure of DNA is a double helix, the model of which was proposed by D. Watson and F. Crick in 1953.

Prerequisites for creating a DNA model

As a result of initial analyzes, the idea was that DNA of any origin contains all four nucleotides in equal molar amounts. However, in the 1940s, E. Chargaff and his colleagues, as a result of the analysis of DNA isolated from various organisms, clearly showed that nitrogenous bases are contained in them in various quantitative ratios. Chargaff found that, although these ratios are the same for DNA from all cells of the same species of organisms, DNA from different species can differ markedly in the content of certain nucleotides. This suggested that the differences in the ratio of nitrogenous bases might be related to some biological code. Although the ratio of individual purine and pyrimidine bases in different DNA samples was not the same, when comparing the results of the analyzes, a certain pattern was revealed: in all samples, the total amount of purines was equal to the total amount of pyrimidines (A + G = T + C), the amount of adenine was equal to the amount of thymine (A = T), and the amount of guanine - the amount of cytosine (G = C). DNA isolated from mammalian cells was generally richer in adenine and thymine and relatively poorer in guanine and cytosine, while DNA from bacteria was richer in guanine and cytosine and relatively poorer in adenine and thymine. These data formed an important part of the factual material, on the basis of which the Watson-Crick DNA structure model was later built.

Another important indirect indication of the possible structure of DNA was L. Pauling's data on the structure of protein molecules. Pauling showed that several different stable configurations of the amino acid chain are possible in a protein molecule. One of the common configurations of the peptide chain - α-helix - is a regular helical structure. With such a structure, the formation of hydrogen bonds between amino acids located on adjacent turns of the chain is possible. Pauling described the α-helical configuration of the polypeptide chain in 1950 and suggested that DNA molecules also probably have a helical structure fixed by hydrogen bonds.

However, the most valuable information about the structure of the DNA molecule was provided by the results of X-ray diffraction analysis. X-rays, passing through a DNA crystal, undergo diffraction, that is, they are deflected in certain directions. The degree and nature of the deflection of the rays depend on the structure of the molecules themselves. The X-ray diffraction pattern (Fig. 3) gives the experienced eye a number of indirect indications regarding the structure of the molecules of the substance under study. Analysis of DNA X-ray diffraction patterns led to the conclusion that the nitrogenous bases (having a flat shape) are stacked like a stack of plates. X-ray patterns made it possible to identify three main periods in the structure of crystalline DNA: 0.34, 2, and 3.4 nm.

Watson-Crick DNA Model

Starting from Chargaff's analytical data, Wilkins' x-rays, and chemists who provided information about the exact distances between atoms in a molecule, about the angles between the bonds of a given atom, and about the size of atoms, Watson and Crick began to build physical models of individual components of the DNA molecule at a certain scale. and "adjust" them to each other in such a way that the resulting system corresponds to various experimental data [show] .

Even earlier, it was known that adjacent nucleotides in a DNA chain are connected by phosphodiester bridges that link the 5'-carbon atom of deoxyribose of one nucleotide to the 3'-carbon atom of deoxyribose of the next nucleotide. Watson and Crick had no doubt that a period of 0.34 nm corresponds to the distance between successive nucleotides in a DNA strand. Further, it could be assumed that the period of 2 nm corresponds to the thickness of the chain. And in order to explain to what real structure the period of 3.4 nm corresponds, Watson and Crick, as well as Pauling earlier, assumed that the chain is twisted in the form of a spiral (or, more precisely, forms a helix, since the spiral in the strict sense of this the word is obtained when the turns form a conical rather than a cylindrical surface in space). Then the period of 3.4 nm will correspond to the distance between successive turns of this spiral. Such a spiral can be very dense or somewhat stretched, i.e., its turns can be flat or steep. Since the period of 3.4 nm is exactly 10 times the distance between consecutive nucleotides (0.34 nm), it is clear that each complete turn of the helix contains 10 nucleotides. From these data, Watson and Crick were able to calculate the density of a polynucleotide chain twisted into a helix with a diameter of 2 nm, with a distance between turns equal to 3.4 nm. It turned out that such a strand would have a density half that of the actual density of DNA, which was already known. I had to assume that the DNA molecule consists of two chains - that it is a double helix of nucleotides.

The next task was, of course, to elucidate the spatial relationship between the two strands forming the double helix. Having tried a number of strand arrangements on their physical model, Watson and Crick found that the best fit for all available data is one in which the two polynucleotide helices run in opposite directions; in this case, chains consisting of sugar and phosphate residues form the surface of a double helix, and purines and pyrimidines are located inside. The bases located opposite each other, belonging to two chains, are connected in pairs by hydrogen bonds; it is these hydrogen bonds that hold the chains together, thus fixing the overall configuration of the molecule.

The DNA double helix can be thought of as a helical rope ladder, with the rungs remaining horizontal. Then two longitudinal ropes will correspond to chains of sugar and phosphate residues, and the crossbars will correspond to pairs of nitrogenous bases connected by hydrogen bonds.

As a result of further study of possible models, Watson and Crick came to the conclusion that each "crossbar" should consist of one purine and one pyrimidine; at a period of 2 nm (corresponding to the diameter of the double helix), there would not be enough room for two purines, and the two pyrimidines could not be close enough together to form proper hydrogen bonds. An in-depth study of the detailed model showed that adenine and cytosine, making up a combination of the right size, still could not be located in such a way that hydrogen bonds formed between them. Similar reports also forced the guanine-thymine combination to be excluded, while the combinations adenine-thymine and guanine-cytosine were found to be quite acceptable. The nature of hydrogen bonds is such that adenine pairs with thymine, and guanine pairs with cytosine. This concept of specific base pairing made it possible to explain the "Chargaff rule", according to which in any DNA molecule the amount of adenine is always equal to the content of thymine, and the amount of guanine is always equal to the amount of cytosine. Two hydrogen bonds form between adenine and thymine, and three between guanine and cytosine. Due to this specificity in the formation of hydrogen bonds against each adenine in one chain, thymine is in the other; in the same way, only cytosine can be placed against each guanine. Thus, the chains are complementary to each other, that is, the sequence of nucleotides in one chain uniquely determines their sequence in the other. The two chains run in opposite directions and their phosphate end groups are at opposite ends of the double helix.

As a result of their research, in 1953 Watson and Crick proposed a model for the structure of the DNA molecule (Fig. 3), which remains relevant to the present. According to the model, a DNA molecule consists of two complementary polynucleotide chains. Each DNA strand is a polynucleotide consisting of several tens of thousands of nucleotides. In it, neighboring nucleotides form a regular pentose-phosphate backbone due to the combination of a phosphoric acid residue and deoxyribose by a strong covalent bond. The nitrogenous bases of one polynucleotide chain are arranged in a strictly defined order against the nitrogenous bases of the other. The alternation of nitrogenous bases in the polynucleotide chain is irregular.

The arrangement of nitrogenous bases in the DNA chain is complementary (from the Greek "complement" - addition), i.e. against adenine (A) is always thymine (T), and against guanine (G) - only cytosine (C). This is explained by the fact that A and T, as well as G and C, strictly correspond to each other, i.e. complement each other. This correspondence is given by the chemical structure of the bases, which allows the formation of hydrogen bonds in a pair of purine and pyrimidine. Between A and T there are two bonds, between G and C - three. These bonds provide partial stabilization of the DNA molecule in space. The stability of the double helix is ​​directly proportional to the number of G≡C bonds, which are more stable than the A=T bonds.

The known sequence of nucleotides in one strand of DNA makes it possible, by the principle of complementarity, to establish the nucleotides of another strand.

In addition, it has been established that nitrogenous bases having an aromatic structure are located one above the other in an aqueous solution, forming, as it were, a stack of coins. This process of forming stacks of organic molecules is called stacking. The polynucleotide chains of the DNA molecule of the considered Watson-Crick model have a similar physicochemical state, their nitrogenous bases are arranged in the form of a stack of coins, between the planes of which van der Waals interactions (stacking interactions) occur.

Hydrogen bonds between complementary bases (horizontally) and stacking interaction between base planes in a polynucleotide chain due to van der Waals forces (vertically) provide the DNA molecule with additional stabilization in space.

The sugar-phosphate backbones of both chains are turned outward, and the bases are inward, towards each other. The direction of the strands in DNA is antiparallel (one of them has the direction 5"->3", the other - 3"->5", i.e. the 3"-end of one strand is located opposite the 5"-end of the other.). The chains form right helixes with a common axis. One turn of the helix is ​​10 nucleotides, the size of the turn is 3.4 nm, the height of each nucleotide is 0.34 nm, the diameter of the helix is ​​2.0 nm. As a result of the rotation of one strand around the other, a major groove (about 20 Å in diameter) and a minor groove (about 12 Å) are formed in the DNA double helix. This form of the Watson-Crick double helix was later called the B-form. In cells, DNA usually exists in the B form, which is the most stable.

Functions of DNA

The proposed model explained many of the biological properties of deoxyribonucleic acid, including the storage of genetic information and the diversity of genes, provided by a wide variety of consecutive combinations of 4 nucleotides and the fact of the existence of a genetic code, the ability to self-reproduce and transmit genetic information, provided by the replication process, and the implementation of genetic information in the form of proteins, as well as any other compounds formed with the help of enzyme proteins.

Basic functions of DNA.

1. DNA is the carrier of genetic information, which is ensured by the fact of the existence of the genetic code.
2. Reproduction and transmitted genetic information in generations of cells and organisms. This function is provided by the replication process.
3. Implementation of genetic information in the form of proteins, as well as any other compounds formed with the help of enzyme proteins. This function is provided by the processes of transcription and translation.

Forms of organization of double-stranded DNA

DNA can form several types of double helixes (Fig. 4). Currently, six forms are already known (from A to E and Z-form).

Structural forms of DNA, as established by Rosalind Franklin, depend on the saturation of the nucleic acid molecule with water. In studies of DNA fibers using X-ray diffraction analysis, it was shown that the X-ray diffraction pattern radically depends on at what relative humidity, at what degree of water saturation of this fiber, the experiment takes place. If the fiber was sufficiently saturated with water, then one radiograph was obtained. When dried, a completely different X-ray pattern appeared, very different from the X-ray pattern of a high-moisture fiber.

Molecule of high humidity DNA is called B-shape. Under physiological conditions (low salt concentration, high degree of hydration), the dominant structural type of DNA is the B-form (the main form of double-stranded DNA is the Watson-Crick model). The helix pitch of such a molecule is 3.4 nm. There are 10 complementary pairs per turn in the form of twisted stacks of "coins" - nitrogenous bases. The stacks are held together by hydrogen bonds between two opposite "coins" of the stacks, and are "coiled" with two ribbons of the phosphodiester backbone twisted into a right-handed helix. The planes of the nitrogenous bases are perpendicular to the axis of the helix. Neighboring complementary pairs are rotated relative to each other by 36°. The helix diameter is 20Å, with the purine nucleotide occupying 12Å and the pyrimidine nucleotide occupying 8Å.

DNA molecule of lower moisture is called A-form. The A-form is formed under conditions of less high hydration and at a higher content of Na + or K + ions. This wider right-handed conformation has 11 base pairs per turn. The planes of nitrogenous bases have a stronger inclination to the axis of the helix, they deviate from the normal to the axis of the helix by 20°. This implies the presence of an internal void with a diameter of 5 Å. The distance between adjacent nucleotides is 0.23 nm, the length of the coil is 2.5 nm, and the diameter of the helix is ​​2.3 nm.

Initially, the A-form of DNA was thought to be less important. However, later it turned out that the A-form of DNA, as well as the B-form, is of great biological importance. The RNA-DNA helix in the template-seed complex has the A-form, as well as the RNA-RNA helix and RNA hairpin structures (the 2'-hydroxyl group of ribose does not allow RNA molecules to form the B-form). The A-form of DNA is found in spores. It has been established that the A-form of DNA is 10 times more resistant to UV rays than the B-form.

The A-form and B-form are called the canonical forms of DNA.

Forms C-E also right-handed, their formation can only be observed in special experiments, and, apparently, they do not exist in vivo. The C-form of DNA has a structure similar to B-DNA. The number of base pairs per turn is 9.33, and the length of the helix is ​​3.1 nm. The base pairs are inclined at an angle of 8 degrees relative to the perpendicular position to the axis. The grooves are close in size to the grooves of B-DNA. In this case, the main groove is somewhat smaller, and the minor groove is deeper. Natural and synthetic DNA polynucleotides can pass into the C-form.

Table 1. Characteristics of some types of DNA structures
Spiral type	A	B	Z
Spiral pitch	0.32 nm	3.38 nm	4.46 nm
Spiral twist	Right	Right	Left
Number of base pairs per turn	11	10	12
Distance between base planes	0.256 nm	0.338 nm	0.371 nm
Glycosidic bond conformation	anti	anti	anti-C syn-G
Furanose ring conformation	C3 "-endo	C2 "-endo	C3 "-endo-G C2 "-endo-C
Groove width, small/large	1.11/0.22 nm	0.57/1.17 nm	0.2/0.88 nm
Groove depth, small/large	0.26/1.30 nm	0.82/0.85 nm	1.38/0.37 nm
Spiral diameter	2.3 nm	2.0 nm	1.8 nm

Structural elements of DNA
(non-canonical DNA structures)

Structural elements of DNA include unusual structures limited by some special sequences:

Z-form of DNA - is formed in places of the B-form of DNA, where purines alternate with pyrimidines or in repeats containing methylated cytosine. Palindromes are flip sequences, inverted repeats of base sequences, having a second-order symmetry with respect to two DNA strands and forming "hairpins" and "crosses". The H-form of DNA and triple helixes of DNA are formed when there is a site containing only purines in one strand of the normal Watson-Crick duplex, and in the second strand, respectively, pyrimidines complementary to them. G-quadruplex (G-4) is a four-stranded DNA helix, where 4 guanine bases from different strands form G-quartets (G-tetrads), held together by hydrogen bonds to form G-quadruplexes.

Z-form of DNA was discovered in 1979 while studying the hexanucleotide d(CG)3 - . It was opened by MIT professor Alexander Rich and his staff. The Z-form has become one of the most important structural elements of DNA due to the fact that its formation was observed in DNA regions where purines alternate with pyrimidines (for example, 5'-HCHCHC-3'), or in repeats 5'-CHCHCH-3' containing methylated cytosine. An essential condition for the formation and stabilization of Z-DNA was the presence in it of purine nucleotides in the syn-conformation, alternating with pyrimidine bases in the anti-conformation.

Natural DNA molecules mostly exist in the right B form unless they contain sequences like (CG)n. However, if such sequences are part of the DNA, then these regions, when the ionic strength of the solution or cations that neutralize the negative charge on the phosphodiester backbone, can change into the Z-form, while other DNA regions in the chain remain in the classical B-form. The possibility of such a transition indicates that the two strands in the DNA double helix are in a dynamic state and can unwind relative to each other, passing from the right form to the left one and vice versa. The biological consequences of this lability, which allows conformational transformations of the DNA structure, are not yet fully understood. It is believed that Z-DNA regions play a role in the regulation of the expression of certain genes and take part in genetic recombination.

The Z-form of DNA is a left-handed double helix, in which the phosphodiester backbone is zigzag along the axis of the molecule. Hence the name of the molecule (zigzag)-DNA. Z-DNA is the least twisted (12 base pairs per turn) and thinnest known in nature. The distance between adjacent nucleotides is 0.38 nm, the coil length is 4.56 nm, and the Z-DNA diameter is 1.8 nm. In addition, the appearance of this DNA molecule is distinguished by the presence of a single groove.

The Z-form of DNA has been found in prokaryotic and eukaryotic cells. To date, antibodies have been obtained that can distinguish between the Z-form and the B-form of DNA. These antibodies bind to specific regions of the giant chromosomes of Drosophila (Dr. melanogaster) salivary gland cells. The binding reaction is easy to follow due to the unusual structure of these chromosomes, in which denser regions (disks) contrast with less dense regions (interdisks). Z-DNA regions are located in the interdiscs. It follows from this that the Z-form actually exists in natural conditions, although the sizes of the individual sections of the Z-form are not yet known.

(shifters) - the most famous and frequently occurring base sequences in DNA. A palindrome is a word or phrase that reads from left to right and vice versa in the same way. Examples of such words or phrases are: HUT, COSSACK, FLOOD, AND A ROSE FALLED ON AZOR'S PAWS. When applied to sections of DNA, this term (palindrome) means the same alternation of nucleotides along the chain from right to left and from left to right (like the letters in the word "hut", etc.).

A palindrome is characterized by the presence of inverted repeats of base sequences having a second-order symmetry with respect to two DNA strands. Such sequences, for obvious reasons, are self-complementary and tend to form hairpin or cruciform structures (Fig.). Hairpins help regulatory proteins to recognize the place where the genetic text of chromosome DNA is copied.

In cases where an inverted repeat is present in the same DNA strand, such a sequence is called a mirror repeat. Mirror repeats do not have self-complementary properties and therefore are not capable of forming hairpin or cruciform structures. Sequences of this type are found in almost all large DNA molecules and can range from just a few base pairs to several thousand base pairs.

The presence of palindromes in the form of cruciform structures in eukaryotic cells has not been proven, although a number of cruciform structures have been found in vivo in E. coli cells. The presence of self-complementary sequences in RNA or single-stranded DNA is the main reason for the folding of the nucleic chain in solutions into a certain spatial structure, which is characterized by the formation of many "hairpins".

H-form of DNA- this is a helix that is formed by three strands of DNA - the triple helix of DNA. It is a complex of the Watson-Crick double helix with the third single-stranded DNA strand, which fits into its large groove, with the formation of the so-called Hoogsteen pair.

The formation of such a triplex occurs as a result of the addition of the DNA double helix in such a way that half of its section remains in the form of a double helix, and the second half is disconnected. In this case, one of the disconnected spirals forms a new structure with the first half of the double helix - a triple helix, and the second turns out to be unstructured, in the form of a single-filament section. A feature of this structural transition is a sharp dependence on the pH of the medium, the protons of which stabilize the new structure. Due to this feature, the new structure was called the H-form of DNA, the formation of which was found in supercoiled plasmids containing homopurine-homopyrimidine regions, which are a mirror repeat.

In further studies, the possibility of structural transition of some homopurine-homopyrimidine double-stranded polynucleotides was established with the formation of a three-stranded structure containing:

• one homopurine and two homopyrimidine strands ( Py-Pu-Py triplex) [Hoogsteen interaction].

  The constituent blocks of the Py-Pu-Py triplex are canonical isomorphic CGC+ and TAT triads. Stabilization of the triplex requires protonation of the CGC+ triad, so these triplexes are dependent on the pH of the solution.

• one homopyrimidine and two homopurine strands ( Py-Pu-Pu triplex) [inverse Hoogsteen interaction].

  The constituent blocks of the Py-Pu-Pu triplex are the canonical isomorphic CGG and TAA triads. An essential property of Py-Pu-Pu triplexes is the dependence of their stability on the presence of doubly charged ions, and different ions are needed to stabilize triplexes of different sequences. Since the formation of Py-Pu-Pu triplexes does not require protonation of their constituent nucleotides, such triplexes can exist at neutral pH.

  Note: the direct and reverse Hoogsteen interaction is explained by the symmetry of 1-methylthymine: a 180 ° rotation leads to the fact that the place of the O4 atom is occupied by the O2 atom, while the system of hydrogen bonds is preserved.

There are two types of triple helixes:

1. parallel triple helixes in which the polarity of the third strand is the same as that of the homopurine chain of the Watson-Crick duplex
2. antiparallel triple helixes, in which the polarities of the third and homopurine chains are opposite.

Chemically homologous chains in both Py-Pu-Pu and Py-Pu-Py triplexes are in antiparallel orientation. This was further confirmed by NMR spectroscopy data.

G-quadruplex- 4-stranded DNA. Such a structure is formed if there are four guanines, which form the so-called G-quadruplex - a round dance of four guanines.

The first hints of the possibility of the formation of such structures were obtained long before the breakthrough work of Watson and Crick - as early as 1910. Then the German chemist Ivar Bang discovered that one of the components of DNA - guanosic acid - forms gels at high concentrations, while other components of DNA do not have this property.

In 1962, using the X-ray diffraction method, it was possible to establish the cell structure of this gel. It turned out to be composed of four guanine residues, linking each other in a circle and forming a characteristic square. In the center, the bond is supported by a metal ion (Na, K, Mg). The same structures can be formed in DNA if it contains a lot of guanine. These flat squares (G-quartets) are stacked to form fairly stable, dense structures (G-quadruplexes).

Four separate strands of DNA can be woven into four-stranded complexes, but this is rather an exception. More often, a single strand of nucleic acid is simply tied into a knot, forming characteristic thickenings (for example, at the ends of chromosomes), or double-stranded DNA forms a local quadruplex at some guanine-rich site.

The most studied is the existence of quadruplexes at the ends of chromosomes - on telomeres and in oncopromoters. However, a complete understanding of the localization of such DNA in human chromosomes is still not known.

All these unusual structures of DNA in the linear form are unstable compared to the B-form of DNA. However, DNA often exists in a ring form of topological tension when it has what is known as supercoiling. Under these conditions, non-canonical DNA structures are easily formed: Z-forms, "crosses" and "hairpins", H-forms, guanine quadruplexes, and the i-motif.

• Supercoiled form - noted when released from the cell nucleus without damage to the pentose-phosphate backbone. It has the form of supertwisted closed rings. In the supertwisted state, the DNA double helix is ​​"twisted on itself" at least once, i.e. it contains at least one supercoil (takes the shape of a figure eight).
• Relaxed state of DNA - observed with a single break (break of one strand). In this case, the supercoils disappear and the DNA takes the form of a closed ring.
• The linear form of DNA is observed when two strands of the double helix are broken.

All three listed forms of DNA are easily separated by gel elecrophoresis.

Tertiary structure of DNA

Tertiary structure of DNA is formed as a result of additional twisting in space of a double-stranded molecule - its supercoiling. Supercoiling of the DNA molecule in eukaryotic cells, in contrast to prokaryotes, is carried out in the form of complexes with proteins.

Almost all eukaryotic DNA is located in the chromosomes of the nuclei, only a small amount of it is found in mitochondria, and in plants and in plastids. The main substance of the chromosomes of eukaryotic cells (including human chromosomes) is chromatin, consisting of double-stranded DNA, histone and non-histone proteins.

Histone proteins of chromatin

Histones are simple proteins that make up up to 50% of chromatin. In all the studied cells of animals and plants, five main classes of histones were found: H1, H2A, H2B, H3, H4, differing in size, amino acid composition and charge (always positive).

Mammalian histone H1 consists of a single polypeptide chain containing approximately 215 amino acids; the sizes of other histones vary from 100 to 135 amino acids. All of them are spiralized and twisted into a globule with a diameter of about 2.5 nm, contain an unusually large amount of positively charged amino acids lysine and arginine. Histones can be acetylated, methylated, phosphorylated, poly(ADP)-ribosylated, and histones H2A and H2B can be covalently linked to ubiquitin. What is the role of such modifications in the formation of the structure and performance of functions by histones has not yet been fully elucidated. It is assumed that this is their ability to interact with DNA and provide one of the mechanisms for regulating the action of genes.

Histones interact with DNA mainly through ionic bonds (salt bridges) formed between the negatively charged phosphate groups of DNA and the positively charged lysine and arginine residues of histones.

Non-histone proteins of chromatin

Non-histone proteins, unlike histones, are very diverse. Up to 590 different fractions of DNA-binding nonhistone proteins have been isolated. They are also called acidic proteins, since acidic amino acids predominate in their structure (they are polyanions). The specific regulation of chromatin activity is associated with a variety of non-histone proteins. For example, enzymes essential for DNA replication and expression can bind to chromatin transiently. Other proteins, say those involved in various regulatory processes, bind to DNA only in specific tissues or at certain stages of differentiation. Each protein is complementary to a specific sequence of DNA nucleotides (DNA site). This group includes:

• a family of site-specific zinc finger proteins. Each "zinc finger" recognizes a specific site consisting of 5 nucleotide pairs.
• a family of site-specific proteins - homodimers. A fragment of such a protein in contact with DNA has a "helix-turn-helix" structure.
• high mobility proteins (HMG proteins - from English, high mobility gel proteins) are a group of structural and regulatory proteins that are constantly associated with chromatin. They have a molecular weight of less than 30 kD and are characterized by a high content of charged amino acids. Due to their low molecular weight, HMG proteins are highly mobile during polyacrylamide gel electrophoresis.
• enzymes of replication, transcription and repair.

With the participation of structural, regulatory proteins and enzymes involved in the synthesis of DNA and RNA, the nucleosome thread is converted into a highly condensed complex of proteins and DNA. The resulting structure is 10,000 times shorter than the original DNA molecule.

Chromatin

Chromatin is a complex of proteins with nuclear DNA and inorganic substances. Most of the chromatin is inactive. It contains densely packed, condensed DNA. This is heterochromatin. There are constitutive, genetically inactive chromatin (satellite DNA) consisting of non-expressed regions, and facultative - inactive in a number of generations, but under certain circumstances capable of expressing.

Active chromatin (euchromatin) is uncondensed, i.e. packed less tightly. In different cells, its content ranges from 2 to 11%. In the cells of the brain, it is the most - 10-11%, in the cells of the liver - 3-4 and kidneys - 2-3%. There is an active transcription of euchromatin. At the same time, its structural organization makes it possible to use the same DNA genetic information inherent in a given type of organism in different ways in specialized cells.

In an electron microscope, the image of chromatin resembles beads: spherical thickenings about 10 nm in size, separated by filamentous bridges. These spherical thickenings are called nucleosomes. The nucleosome is the structural unit of chromatin. Each nucleosome contains a 146 bp long supercoiled DNA segment wound to form 1.75 left turns per nucleosome core. The nucleosomal core is a histone octamer consisting of histones H2A, H2B, H3 and H4, two molecules of each type (Fig. 9), which looks like a disk with a diameter of 11 nm and a thickness of 5.7 nm. The fifth histone, H1, is not part of the nucleosomal core and is not involved in the process of DNA winding around the histone octamer. It contacts DNA at the points where the double helix enters and exits the nucleosomal core. These are intercore (linker) sections of DNA, the length of which varies depending on the type of cell from 40 to 50 nucleotide pairs. As a result, the length of the DNA fragment that is part of the nucleosomes also varies (from 186 to 196 nucleotide pairs).

The nucleosome contains about 90% of DNA, the rest of it is the linker. It is believed that nucleosomes are fragments of "silent" chromatin, while the linker is active. However, nucleosomes can unfold and become linear. Unfolded nucleosomes are already active chromatin. This clearly shows the dependence of the function on the structure. It can be assumed that the more chromatin is in the composition of globular nucleosomes, the less active it is. Obviously, in different cells the unequal proportion of resting chromatin is associated with the number of such nucleosomes.

On electron microscopic photographs, depending on the conditions of isolation and the degree of stretching, chromatin can look not only as a long thread with thickenings - "beads" of nucleosomes, but also as a shorter and denser fibril (fiber) with a diameter of 30 nm, the formation of which is observed during the interaction histone H1 associated with the linker region of DNA and histone H3, which leads to additional twisting of the helix of six nucleosomes per turn with the formation of a solenoid with a diameter of 30 nm. In this case, the histone protein can interfere with the transcription of a number of genes and thus regulate their activity.

As a result of the interactions of DNA with histones described above, a segment of the DNA double helix of 186 base pairs with an average diameter of 2 nm and a length of 57 nm turns into a helix with a diameter of 10 nm and a length of 5 nm. With the subsequent compression of this helix to a fiber with a diameter of 30 nm, the degree of condensation increases by another six times.

Ultimately, the packaging of the DNA duplex with five histones results in a 50-fold DNA condensation. However, even such a high degree of condensation cannot explain the almost 50,000-100,000-fold DNA compaction in the metaphase chromosome. Unfortunately, the details of the further packing of chromatin up to the metaphase chromosome are not yet known; therefore, only general features of this process can be considered.

Levels of DNA compaction in chromosomes

Each DNA molecule is packaged into a separate chromosome. Diploid human cells contain 46 chromosomes, which are located in the cell nucleus. The total length of the DNA of all the chromosomes of a cell is 1.74 m, but the diameter of the nucleus in which the chromosomes are packed is millions of times smaller. Such a compact packing of DNA in chromosomes and chromosomes in the cell nucleus is provided by a variety of histone and non-histone proteins interacting in a certain sequence with DNA (see above). Compaction of DNA in chromosomes makes it possible to reduce its linear dimensions by about 10,000 times - conditionally from 5 cm to 5 microns. There are several levels of compactization (Fig. 10).

• DNA double helix is ​​a negatively charged molecule with a diameter of 2 nm and a length of several cm.
• nucleosomal level- chromatin looks in an electron microscope as a chain of "beads" - nucleosomes - "on a thread". The nucleosome is a universal structural unit that is found both in euchromatin and heterochromatin, in the interphase nucleus and metaphase chromosomes.

  The nucleosomal level of compaction is provided by special proteins - histones. Eight positively charged histone domains form the core (core) of the nucleosome around which the negatively charged DNA molecule is wound. This gives a shortening by a factor of 7, while the diameter increases from 2 to 11 nm.

• solenoid level

  The solenoid level of chromosome organization is characterized by the twisting of the nucleosomal filament and the formation of thicker fibrils 20-35 nm in diameter from it - solenoids or superbids. The solenoid pitch is 11 nm, and there are about 6-10 nucleosomes per turn. Solenoid packing is considered more probable than superbid packing, according to which a chromatin fibril with a diameter of 20–35 nm is a chain of granules, or superbids, each of which consists of eight nucleosomes. At the solenoid level, the linear size of DNA is reduced by 6-10 times, the diameter increases to 30 nm.

• loop level

  The loop level is provided by non-histone site-specific DNA-binding proteins that recognize and bind to specific DNA sequences, forming loops of approximately 30-300 kb. The loop ensures gene expression, i.e. the loop is not only a structural, but also a functional formation. Shortening at this level occurs by 20-30 times. The diameter increases to 300 nm. Loop-like "lampbrush" structures in amphibian oocytes can be seen on cytological preparations. These loops appear to be supercoiled and represent DNA domains, probably corresponding to units of chromatin transcription and replication. Specific proteins fix the bases of the loops and, possibly, some of their internal regions. The loop-like domain organization facilitates the folding of chromatin in metaphase chromosomes into helical structures of higher orders.

• domain level

  The domain level of chromosome organization has not been studied enough. At this level, the formation of loop domains is noted - structures of filaments (fibrils) 25-30 nm thick, which contain 60% protein, 35% DNA and 5% RNA, are practically invisible in all phases of the cell cycle with the exception of mitosis and are somewhat randomly distributed over cell nucleus. Loop-like "lampbrush" structures in amphibian oocytes can be seen on cytological preparations.

  Loop domains are attached with their base to the intranuclear protein matrix in the so-called built-in attachment sites, often referred to as MAR / SAR sequences (MAR, from the English matrix associated region; SAR, from the English scaffold attachment regions) - DNA fragments several hundred long base pairs that are characterized by a high content (>65%) of A/T base pairs. Each domain appears to have a single origin of replication and functions as an autonomous supercoiled unit. Any loop domain contains many transcription units, the functioning of which is likely to be coordinated - the entire domain is either in an active or inactive state.

  At the domain level, as a result of sequential packing of chromatin, the linear dimensions of DNA decrease by about 200 times (700 nm).

• chromosome level

  At the chromosomal level, the prophase chromosome condenses into a metaphase one with the compaction of loop domains around the axial framework of non-histone proteins. This supercoiling is accompanied by phosphorylation of all H1 molecules in the cell. As a result, the metaphase chromosome can be depicted as densely packed solenoid loops coiled into a tight spiral. A typical human chromosome can contain up to 2600 loops. The thickness of such a structure reaches 1400 nm (two chromatids), while the DNA molecule is shortened by 104 times, i.e. from 5 cm stretched DNA to 5 µm.

Functions of chromosomes

In interaction with extrachromosomal mechanisms, chromosomes provide

1. storage of hereditary information
2. using this information to create and maintain cellular organization
3. regulation of reading hereditary information
4. self-duplication of genetic material
5. the transfer of genetic material from a mother cell to daughter cells.

There is evidence that upon activation of a chromatin region, i.e. during transcription, histone H1 is reversibly removed from it first, and then the histone octet. This causes decondensation of chromatin, successive transition of a 30-nm chromatin fibril into a 10-nm filament, and its further unfolding into regions of free DNA, i.e. loss of nucleosomal structure.

Behavior: an evolutionary approach Kurchanov Nikolai Anatolievich

1.2. Organization of genetic material

The structural and functional organization of the genetic apparatus determines the division of all living organisms into prokaryotes and eukaryotes. In prokaryotes (which include bacteria and archaea), DNA is represented by a circular molecule and is located in the cytoplasm of the cell. In eukaryotes (which include all other organisms), DNA is the structural carrier of genetic information. chromosomes, located in the nucleus.

Chromosomes are a complex multilevel structure in which DNA interacts with various proteins. The base level of this structure is nucleosomes which are globules of eight protein molecules histones, entwined DNA. The nucleohistone strand is further folded many times, forming compact chromosomes. This structure opens up wide possibilities for regulation.

Since the number of genes in an organism is incommensurably greater than the number of chromosomes, it is clear that each chromosome carries many genes. Each gene occupies a specific place on the chromosome. locus. Genes located on the same chromosome are called linked.

In addition to the nucleus, a small proportion of the genetic information of a eukaryotic cell is located in organelles such as mitochondria and chloroplasts, which have their own genetic systems: their own DNA, various RNAs (i-RNA, t-RNA, r-RNA) and ribosomes, which allows independent synthesis squirrel. The circular DNA of these organelles was an important argument in favor of their bacterial symbiotic origin at the dawn of the formation of life.

The cell nucleus of eukaryotes separates the processes of transcription and translation, which provides ample opportunities for regulation. Regulation occurs at all stages of eukaryotic gene expression. Their additional step is processing - the process of complex transformations of RNA synthesized during transcription. The most important component of mRNA processing is splicing, at which cutting takes place introns(non-coding regions of the gene) and cross-linking exons(coding regions). Exons and introns determine the "mosaic" structure of eukaryotic genes. It is as a result of processing that the RNA synthesized in the nucleus becomes functionally active.

Understanding the diverse mechanisms of regulation has caused radical changes in our understanding of the structural and functional organization of the genetic apparatus at the present time.

One of the founders of modern genetics, the outstanding Danish scientist W. Johannsen (1857–1927), proposed basic genetic terms - gene, allele, genotype, phenotype, which determine the genetic characteristics of an individual.

Genes located at their loci can have variants − alleles. A locus that has more than one allele in a population is called polymorphic. Usually, alleles are denoted by letters of the Latin or Greek alphabet, and if there are many of them, then with a superscript. The number of alleles of different genes in populations of organisms can be different. Some genes have many alleles, others have few. In any case, the number of alleles is limited by evolutionary factors: alleles that impair the adaptive properties of the species or are incompatible with life are eliminated by natural selection.

A particular eukaryotic organism has only two alleles of one gene: according to the number of homologous loci of homologous chromosomes (paternal and maternal). An organism in which both alleles are the same is called homozygous(for this gene). An organism that has different alleles is called heterozygous(Fig. 1.4). Alleles localized on the sex chromosomes of the heterogametic sex may be present in the singular.

Genotype can be represented as a set of alleles of an organism, and phenotype - as a set of its external features.

Introduced in 1920 by the German botanist G. Winkler (1877–1945), the term genome became a characteristic of a whole species of organisms, and not a specific individual. This concept later became one of the most important. By the 1980s 20th century a new branch of genetics, genomics, is emerging. Initially, the genome was characterized as a collection of haploid gene loci. However, it turned out that the genes themselves occupy a relatively small part of the genome, although they form its basis. Most of them are occupied by intergenic regions, where there are regions with a regulatory function, as well as regions of an unknown destination. Regulatory regions are inextricably linked with genes, they are a kind of "instructions" that determine the work of genes at different stages of development of the organism. Therefore, the genome is currently called the entire set of DNA of the cell, characteristic of the DNA of the species.

At the present stage of development of genetics, genomics is becoming one of its key sections. The success of genomics was clearly demonstrated by the successful completion of the Human Genome Program.

Rice. 1.4. Alleles of linked genes of two homologous chromosomes

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Chapter B. Baryon Digitization of the Genetic Code (xiv)

According to the chemical organization of the material of heredity and variability, eukaryotic and prokaryotic cells do not fundamentally differ from each other. Their genetic material is represented by DNA. Common to them is the principle of recording genetic information, as well as the genetic code. The same amino acids are encrypted in pro- and eukaryotes with the same codons. In principle, the use of hereditary information stored in DNA is carried out in the same way in these types of cells. However, some features of the organization of hereditary material, which distinguish eukaryotic cells from prokaryotic ones, cause differences in the use of their genetic information.

The hereditary material of a prokaryotic cell is contained mainly in a single circular DNA molecule.

The hereditary material of eukaryotes is larger in volume than that of prokaryotes. It is located mainly in chromosomes that are separated from the cytoplasm by the nuclear envelope.

Significant differences exist in the molecular organization of genes in eukaryotic cells. Most of them have coding sequences exons interrupted intron sites that are not used in the synthesis of tRNA, rRNA or peptides. These regions are removed from the primary transcribed RNA, and therefore the use of genetic information in a eukaryotic cell occurs somewhat differently. In a prokaryotic cell, where the hereditary material and the apparatus for protein biosynthesis are not spatially separated, transcription and translation occur almost simultaneously. In a eukaryotic cell, these two stages are not only spatially separated by the nuclear envelope, but they are also separated in time by the processes of mRNA maturation, from which uninformative sequences must be removed.

Chemical organization of the genetic material.

gene level.

The elementary functional unit of the genetic apparatus, which determines the possibility of developing an individual trait of a cell or organism of a given species, is gene(hereditary deposit, according to G. Mendel). By transferring genes in a series of generations of cells or organisms, material continuity is achieved - the inheritance of parental traits by descendants.

Under sign understand the unit of morphological, physiological, biochemical, immunological, clinical and any other discreteness of organisms (cells), i.e. a separate quality or property by which they differ from each other.

chromosome level.

The genes of eukaryotic cells are distributed over chromosomes, forming the CHROMOSOMAL level of organization of hereditary material. This level of organization serves as a necessary condition for the linkage of genes and the redistribution of parental genes in offspring during sexual reproduction (crossing over).

Chromosomes- nucleoprotein structures in the nucleus of a eukaryotic cell, in which most of the hereditary information is concentrated and which are designed for its storage, implementation and transmission.

genomic level.

Genome - the entire set of hereditary material contained in the haploid set of chromosomes of cells of a given type of organism. The genome is species-specific, since it is the necessary set of genes that ensures the formation of species characteristics of organisms during their normal ontogenesis.

The structure of the gene.

Studies aimed at elucidating the chemical nature of hereditary material have irrefutably proved that the material substrate of heredity and variability are nucleic acids, which were discovered by F. Miescher (1868) in the nuclei of pus cells. Nucleic acids are macromolecules, i.e. have a high molecular weight. These are polymers that are made up of monomers. nucleotides including three components: sugar(pentose), phosphate and nitrogenous base(purine or pyrimidine). A nitrogenous base (adenine, guanine, cytosine, thymine or uracil) is attached to the first carbon atom in the C-1′ pentose molecule, and a phosphate is attached to the fifth carbon atom C-5′ using an ether bond; the third carbon atom C-3' always has a hydroxyl group - OH.

The connection of nucleotides into a nucleic acid macromolecule occurs by the interaction of the phosphate of one nucleotide with the hydroxyl of another so that between them is established phosphodiester bond. The result is a polynucleotide chain. The backbone of the chain consists of alternating phosphate and sugar molecules. One of the nitrogenous bases listed above is attached to the pentose molecules in the C-1' position.

DNA structure, properties and functions.

DNA consists of nucleotides, which include sugar - deoxyribose, phosphate and one of the nitrogenous bases - adenine, guanine, thymine, cytosine. DNA molecules include two polynucleotide chains linked together in a certain way. Watson and Crick suggested that these chains are connected to each other by hydrogen bonds between their nitrogenous bases according to the principle of complementarity. Adenine of one chain is connected by two hydrogen bonds with Thymine of another chain, and three hydrogen bonds are formed between guanine and cytosine of different chains. Such a connection of nitrogenous bases provides a strong connection between the two chains and maintaining an equal distance between them throughout. Another important feature of two polynucleotide chains in a DNA molecule is their antiparallelism: the 5-end of one chain is connected to the 3-end of the other and vice versa. X-ray diffraction data showed that a DNA molecule, consisting of two chains, forms a helix twisted around its axis. Helix diameter 2 nm, pitch length 3.4 nm. Each turn contains 10 pairs of nucleotides. That. in the structural organization of the DNA molecule, one can distinguish the primary structure - a polynucleotide chain, the secondary - two complementary and antiparallel chains, and the tertiary

The structure is a three-dimensional spiral.

DNA is capable of self-copying - replication. In the process of replication, a complementary chain is synthesized on each polynucleotide chain of the parent DNA molecule. As a result, two identical double helixes are formed from one DNA double helix. Such a way of doubling molecules, in which each daughter molecule has one parent and one newly synthesized chain, is called semi-conservative. For replication to occur, maternal DNA must be separated from each other to become templates on which complementary strands of daughter molecules will be synthesized. With the help of the helicase enzyme, the double helix of DNA in separate zones unwinds. The resulting single-stranded regions are bound by special destabilizing proteins. The molecules of these proteins line up along the polynucleotide chains, stretching their backbone and making nitrogenous bases available for binding to complementary nucleotides. Areas of divergence of polynucleotide chains in replication zones are called replication forks. In each such region, with the participation of the DNA polymerase enzyme, the DNA of two new daughter molecules is synthesized. In the process of synthesis, the replication fork moves along the parent helix, capturing all new zones. The end result of replication is the formation of two DNA molecules whose nucleotide sequence is identical to that of the parent DNA double helix,