Textbook - BIOLOGICAL CHEMISTRY - Hubsky Y.I. - 2000

Chapter IV. MOLECULAR MECHANISMS OF HEREDITY AND THE REALIZATION OF GENETIC INFORMATION

CHAPTER 22. REGULATION OF GENE EXPRESSION. GENETIC RECOMBINATIONS

22.2. FEATURES OF THE MOLECULAR ORGANIZATION AND EXPRESSION OF THE EUKARYOTIC GENOME

Data from modern biochemistry and molecular biology indicate that the composition and subcellular architecture of The Introduction/5.html">Eukaryotic Cell differ significantly from prokaryotes: for instance, the number of individual Proteins in The Human Body exceeds 50,000, compared to approximately 3,000 different proteins in E. coli. Accordingly, The Genome of nuclear organisms is a much more complex system in its Structure and molecular Organization than that of prokaryotes, with notable differences existing in quantitative parameters and the Structural organization of DNA.

Due to the specific structural ORGANIZATION OF THE genome, the Control of Gene Expression in eukaryotes is also considerably more complex. In addition to mechanisms similar to those found in prokaryotes, gene expression control in Eukaryotic Cells involves specific molecular processes that exert regulatory effects at various levels of gene expression:

(1) at the level of the structural organization of the genome — regulation is achieved through the presence of specific nucleotide sequences, gene rearrangements (gene recombination), and gene Amplification;

(2) at the METABOLISM/31.html">Transcription level — regulatory mechanisms include The Influence of transcription enhancement and attenuation signals (enhancers and attenuators, respectively) and post-transcriptional mRNA modification;

(3) at the Translation level — the primary regulatory mechanism is the Covalent Modification of protein translation factors through their reversible phosphorylation-dephosphorylation.

Class="center">Molecular Organization of Eukaryotic DNA

The haploid genome of each cell in a Homo sapiens Organism contains 3.5 · 10⁹ Base Pairs and consists of 23 pairs of Chromosomes (Fig. 22.7), which is sufficient to form about 1.5 million gene pairs. At the same time, the human body actually synthesizes no more than 100,000 different proteins; in other words, the greater part of the nuclear DNA that makes up The Human Genome is not translated into the Amino acid sequences of protein molecules.

Fig. 22.7. Structure of human chromosome 12 (x 27,850).

The presence (in addition to regulatory and signaling nucleotide sequences also present in prokaryotes) of a significant number of non-transcribed regions is a specific feature of the genome structure in eukaryotic cells. Such "silent" genomic fragments are termed introns, in contrast to exons — genomic regions that are transcribed to form mRNAs carrying information for the Synthesis of specific cellular proteins.

According to current estimates, only 2% of mammalian cellular DNA contains information encoding the organism's proteins (L. Stryer, 1995).

Repetitive DNA Sequences

Studies of DNA nucleotide sequences in higher organisms have revealed that, alongside sequences with unique nucleotide compositions, There is a high prevalence of fragments present in mammalian DNA in many copies — ranging from 2 to 10⁷ repeats per cell. In the human genome, repeats account for 20-30% of its total length.

Depending on the number of NUCLEOTIDES and their repetition frequency, repetitive nucleotide sequences are classified into the following groups:

(а) highly repetitive sequences, which are regions ranging from 5 to 500 base pairs in length, arranged consecutively ("in tandem"). These sequences form clusters containing 1 to 10 million copies and constitute the "satellite DNA" fraction. These highly repetitive sequences are transcriptionally inactive, and their functional role remains not fully understood;

(б) moderately repetitive sequences, which have no more than 10⁶ copies and do not form tandem clusters, but instead alternate with unique nucleotide sequences ("dispersed repeats"). These sequences vary in length and can be either short or long (5–7 thousand base pairs).

Short interspersed repeats are DNA fragments ranging from a few base pairs to several hundred base pairs in length. This class includes the Alu family repeats, which are widespread in human cells, have a length of 300 base pairs, and are repeated in approximately 500,000 copies, altogether making up 3–6% of the total haploid Genome Size. These repeats can be transcribed as components of hnRNA and individual cellular RNAs, such as 4.5S and 7S RNAs.

In their nucleotide structure, Alu repeats are similar to the LTR terminal sequences of Retroviruses and likely share a genetic connection with them. It is believed that these sequences are Mobile Genetic Elements capable of transposition, meaning excision and insertion into various Regions of the genome.

Eukaryotic Nuclear Chromatin

As noted previously, a structural feature of the Spatial Organization of eukaryotic DNA is its compaction into supercoils which, in complex with Histones and non-histone proteins (NHPs — which include regulatory and structural proteins, Replication Enzymes, etc.), form nuclear chromatin. During mitosis, this chromatin organizes into discrete bodies known as chromosomes.

In animal and human cells, DNA replication occurs exclusively during a specific period of the Cell Cycle — the S phase, which is separated from mitosis (M) by the pre-synthetic (G₁) and post-synthetic (G₂) phases. The S, G₁, and G₂ phases constitute the intermitotic period, or interphase, while G₀ represents a period of reproductive quiescence (Fig. 22.8). The control mechanisms regulating The Cell's transition from G₁ to the S phase remain insufficiently understood. However, the triggering reactions that determine the onset of mitosis, which involve the reversible phosphorylation of specific nuclear and cytoplasmic proteins, have been largely elucidated in recent years (see below).

Fig. 22.8. Phases of the cell cycle.

The nucleosomal organization of nuclear chromatin and the Biochemical characteristics of histones were discussed in Chapter 3. In the interphase nucleus, chromatin is differentiated into transcriptionally active chromatin (TAC), or "euchromatin", and transcriptionally inactive, repressed chromatin (RC), or "heterochromatin". These Two Types of chromatin are distinguished by their biochemical and morphological (electron microscopic) features: TAC is less tightly compacted than RC, its nucleosomal structure is altered, and it is entirely absent in the most transcriptionally active regions.

Covalent Modification of Histones and NHPs

Histones and non-histone proteins (NHPs) that make up chromatin nucleosomes undergo post-translational covalent modification. This alters their chemical properties—specifically their ability to interact with specific DNA regions—and may serve as one of the biochemical mechanisms controlling the expression level of specific genes.

Reactions of covalent modification of nuclear chromatin proteins include their Acetylation, phosphorylation, methylation, glycosylation, and ADP-ribosylation. Non-histone proteins undergo post-synthetic modifications to a much greater extent than histones. The respective radicals (acetyl, phosphoryl, methyl, glycosyl, ADP-ribosyl) attach to the side chains of amino acid residues within The polypeptide chains of histones and NHPs.

It should be acknowledged that the regulatory significance of these nuclear protein covalent modification pathways in driving specific molecular-genetic processes has not been definitively established for any of them. Clear functional correlations with genome activity have been found only for acetylation and phosphorylation.

Histone acetylation is a process that begins already in the Cytoplasm (acetylation of the N-terminal Serine of histone H4) and concludes in The Nucleus, where it predominantly targets the side chains of Lysine residues. This leads to a decrease in the positive charge of histones and the loosening of histone-DNA complexes within nucleosomes. Acetylation precedes genome activation and an increase in transcription rate.

Phosphorylation-dephosphorylation of chromatin proteins (specifically the hydroxyl groups of Serine and Threonine residues) is a class of reactions occurring both in the cytoplasm (immediately following translation) and in the nucleus. Studies show that throughout various phases of the cell cycle, both histones (H1, H3) and certain NHPs undergo reversible phosphorylation, though these patterns may vary for individual proteins.

Genetic Recombination

An important role in the Genome Organization of various cells is played by gene rearrangements, or genetic recombination (gene recombination), which refers to the exchange of DNA fragments between different genes or the combination of Genes from different biological sources to form novel chromosomal structures capable of replication and genetic expression (transcription and translation).

Genetic recombination occurs in biological systems of varying complexity—from Viruses and Bacteriophages to higher eukaryotes. Gene structural rearrangements serve as a driving force of Variability and are critically important both for establishing biochemical individuality at the individual organism level within a species and for speciation during evolution.

The MOLECULAR MECHANISMS OF genetic recombination are complex and differ significantly among organisms and types of recombination. They involve enzymatic processes of "cutting" recipient DNA molecules (mediated by specific DNAses) and integrating foreign polynucleotide fragments from another chromosome or from another locus of the same chromosome—so-called Transposons (from the English "transpose"). The ability of transposons to integrate into other DNA molecules depends on specialized nucleotide sequences located at their ends, known as insertion sequences (from the English insert); subsequent ligation of the DNA fragments by DNA ligases completes the gene transfer process (Fig. 22.9).

Fig. 22.9. Insertion of a transposon into recipient DNA. Insertion sequences (IS elements) are located at both ends of the transposon.

Recombination in Prokaryotes

Recombination is a widespread process in prokaryotic organisms, enabling cells to incorporate DNA fragments (genetic elements) from other cells into their own genome. Genetic recombination in prokaryotes includes:

a) transformation—the process by which a recipient microorganism incorporates DNA from a lysed donor cell of the same species;

b) Transduction—The transfer of a DNA fragment from an infected cell into the genome of another recipient microorganism via a bacteriophage;

c) conjugation—a process of sexual reproduction occurring in certain bacterial species, involving the transfer of a DNA fragment from a donor (F⁺) to a recipient (F^-) cell.

Recombination in Eukaryotes

In higher organisms, genetic recombination is a vital component in The formation of haploid egg and sperm cells during Meiosis. Prior to the reductional Cell Division, homologous chromosomes exchange DNA segments (Crossing Over), which serves as the molecular-genetic mechanism allowing offspring to inherit traits from both parental organisms.

Immunoglobulin Gene Recombination

In mature organisms, the translocation or transposition of individual genes or gene groups to another Location within the genome (either within the same chromosome or to a different chromosome) occurs in B-lymphocyte genes encoding IMMUNOGLOBULINS. These genetic recombination events provide the mechanism that enables the Human and Animal body to synthesize millions of different Antibodies in response to invading foreign Antigens (Chapter 30).

Immunoglobulins are tetrameric proteins consisting of four polypeptide chains: two identical H ("heavy") chains and two identical L ("light") chains. There are five types of H-chains (α — alpha, γ — gamma, μ — mu, δ — delta, ε — epsilon) and two types of L-chains (k — kappa and λ — lambda). Depending on the combination of light and heavy chains, five classes of immunoglobulins are distinguished (IgG, IgA, IgM, IgD, and IgE), in each of which a specific type of H-chain pairs with one of the two types of L-chain.

In turn, within each of the H and L chains of immunoglobulin molecules, distinct Structural domains can be identified: constant (C) regions, located at the C-termini of the H and L chains and characterized by a constant Amino Acid Composition across different immunoglobulin classes, and variable (V) regions, located at the N-termini of the H and L chains, which exhibit a variable amino acid composition, forming Conformations complementary to antigen determinant groups and thus ensuring their binding to antibodies.

Fig. 22.10. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF an immunoglobulin molecule.

It is precisely due to the presence of variable regions at the ends of the L and H chains of immunoglobulins that an exceptionally vast number of individual immunoglobulins (antibodies) can be synthesized in response to the introduction of foreign protein molecules. In turn, the synthesis of such a significant number of molecules with diverse primary structures is determined by multiple recombinations of genes encoding individual parts of immunoglobulin molecules.

Let us examine the fundamental patterns of genetic recombination involved in the formation of genes encoding L and H immunoglobulin chains in mature lymphocytes.

L chains — the synthesis of light chains occurs through the expression of three gene families forming the variable (V_L), joining (J_L), and constant (C_L) segments; furthermore, these three families encoding kappa-type light chains are localized on chromosome 2, while those encoding lambda-type chains are on chromosome 22.

The haploid chromosome set contains about 500 GENES OF THE V_L family, 56 genes of the J_L type, and 10–20 genes of the C_L type. These gene families are located at a distance from one another in different regions of the chromosomes (2 and 22, respectively) and are brought together during the maturation of a B lymphocyte through DNA rearrangement and the translocation of a V_L-type gene from a distal chromosomal region closer to the J_L and C_L segments. This rearrangement results in a single gene locus with the structure V_L(i)J_L(i)C_L(i), which is transcribed as a single polycistronic pre-mRNA (primary transcript) that subsequently undergoes Processing to yield the mature mRNA for the immunoglobulin light chain.

Fig. 22.11. Gene families forming immunoglobulin H chains (Stryer L., 1995).

H chains — the synthesis of immunoglobulin heavy chains is encoded by four gene families that form the V_H, D, J_H, and C_H segments located on chromosome 14:

The genetic locus encoding the variable region of the heavy chain is formed by bringing together genes from the V_H (250–350 genes), D (15–20 genes), and J_H (4 genes) families. The genetic Determination of the synthesis of an intact heavy chain occurs through the interaction of the V_H(i)D_(i)J_H(i) structural locus (which encodes the variable region) with one of the eight genes of the C_H family (which encode the constant region).

The synthesis by lymphocytes of specific immunoglobulin classes (G, A, M, D, E) that differ in heavy chain structure is ensured by The Mechanism of "heavy chain class switching," which is executed by fixing a specific chosen V_H gene with a specific C_H gene within the genome structure. Such gene rearrangements underlie the synthesis of immunoglobulin heavy chains during the differentiation of embryonic lymphocytes (A.Ya. Nikolaev, 1998).

Thus, owing to the potential combinations of three gene families (V_L, J_L, and C_L) during light chain synthesis and four gene families (V_H, D, J_H, and C_H) during heavy chain synthesis, The Immune System generates clones of lymphocytes with a diverse gene library that enable the expression of a vast repertoire of immunoglobulins (up to 10⁷–10⁸) with varying antigenic Specificity and functional properties.

Gene Amplification

Gene amplification is The process of increasing the copy number of specific genes.

The Molecular Basis of amplification is the repeated ("explosive") initiation of DNA Synthesis (replication) at the same replication site (Fig. 22.12).

Fig. 22.12. Schematic diagram of gene amplification.

Examples of gene amplification in higher organisms include:

(1) amplification of metallothionein genes — a protein that binds heavy Metal Ions toxic to mammalian organisms, such as mercury, copper, zinc, and cadmium. When these ions enter the body, the transcription frequency of the metallothionein gene increases dozens of times, serving as a physiological mechanism for heavy metal detoxification;

(2) amplification of the Dihydrofolate Reductase gene — an enzyme that converts Folic acid into its coenzyme forms, dihydrofolates, which act as Coenzymes in purine and thymine synthesis.

Amplification of the dihydrofolate reductase gene and a hundreds-fold increase in the level of enzyme synthesis are observed upon administration of the drug methotrexate to Cancer patients. The antitumor action of methotrexate is based on the inhibition of dihydrofolate reductase activity, which disrupts nucleic acid synthesis in malignant tumor cells. Consequently, this upregulated enzyme synthesis driven by gene amplification results in the loss of target cell sensitivity to the antitumor medication.

Polymerase Chain Reaction

The phenomenon of gene amplification has found vital Practical Application in the polymerase chain reaction (PCR) method, which was proposed in 1983 by the American researcher K. Mullis and has become widespread in modern biomedical research.

The PCR method allows obtaining in pure form and in significant quantities (sufficient for research) specific fragments (sequences) of DNA from the complex human genome and identifying them by their nucleotide composition. The Principle of the method is based on The Use of RNA primers specific to certain regions of the analyzed DNA, followed by the use of DNA polymerase, which is capable of replicating nucleotide sequences in both complementary strands, thereby generating A large number of copies of the specific polynucleotide fragment under investigation. The amplified DNA fragments are isolated from the reaction medium and studied using existing Biochemical Methods.

PCR enables so-called "DNA Diagnostics," which is the analysis of specific DNA sequences in human cells. This is of paramount importance in diagnosing Hereditary diseases, detecting the presence of certain viruses in the human body (including HIV), and personal identification. Notably, it was this method that was used for the forensic medical Analysis of the remains of the Romanov imperial family and the identification of the body remains of the Russian Tsar Nicholas II.

Regulation of EUKARYOTIC GENE EXPRESSION at the Transcriptional Level

Genes of higher organisms possess a well-developed system of transcription regulation signals that not only indicate the initiation site of RNA Synthesis but also modulate its activity. The system of transcription signals in eukaryotes includes:

(1) promoters specific to various RNA polymerases, yet sharing a common nucleotide sequence (TATA...) homologous to the Pribnow box in prokaryotes; RNA polymerase II interacts with this nucleotide block;

(2) specific template DNA sequences that enhance or attenuate the level of structural gene expression by influencing transcription activity—specifically, the number of mRNA molecules synthesized and their formation rate—known as enhancer, attenuator, silencer, and adaptor elements of the genome.

Enhancers

Currently, the most thoroughly studied activating elements of the transcription regulation system are positive regulators known as enhancers. Enhancers are DNA regions that can consist of tandemly repeated nucleotide sequences (several tens of base pairs in length, referred to as "modules" or "motifs").

Enhancers increase the transcription efficiency of the genes they influence by tens or hundreds of times. The nucleotide sequences of enhancers have been established for many enzyme proteins (Chymotrypsin, Alcohol dehydrogenase, etc.), Hormones (Insulin, human placental lactogen), and immunoglobulins.

Enhancers are capable of affecting The activity of target genes even when located several thousand base pairs away from their promoters. The activating effect of enhancers is mediated by regulatory proteins (see below) that interact with them and can influence the transcription of distant DNA regions. At the same time, upon interaction with certain protein factors, some enhancers can acquire The properties of negative regulators—silencers—which inhibit the expression of corresponding genes.

(3) numerous regulatory proteins that serve as Components of the transcription signal system, controlling the synthesis activity of various classes of mRNA. To date, proteins capable of specific interactions with particular enhancers have been isolated. These proteins can interact with regulatory DNA sequences located thousands of base pairs away from the RNA polymerase binding and Transcription initiation sites. It is believed that such distal effects of regulatory proteins are mediated by Changes in the spatial conformation of DNA strands and the formation of internal loops that bring regulatory genomic elements close to the transcribed regions.

Steroid Hormones OF the adrenal cortex play a crucial role in the adaptation of higher animals to changing environmental conditions. It has been established that the action of these physiologically active compounds is mediated by specific proteins—receptors—which, in a complex with hormones, interact with specific enhancer regions of the genome, thereby activating the Transcription of DNA from particular genes.

Regulation of Eukaryotic Gene Expression at the Translational Level

The mechanism regulating Protein Biosynthesis at the translational level involves the covalent modification of translational protein factors through reversible phosphorylation-dephosphorylation mediated by a cAMP-dependent cellular regulatory cascade.

Translational control via phosphorylation (inactivation) and dephosphorylation (activation) of the Translation initiation factor eIF-2 (illustrated by The regulation of Hemoglobin synthesis) was discussed in Chapter 21.

Control of Cell Entry into Mitosis

DNA replication in eukaryotes occurs during the S phase of the cell cycle, whereas mitosis—the distribution of duplicated chromosomal material between daughter cells—begins only after the preparatory G₂ phase. Entry into the M phase is preceded by a complex reorganization of cellular architecture, which includes chromatin Condensation, nuclear envelope breakdown, and Cytoskeleton reorganization with the Formation of the mitotic spindle, among other processes.

The key event in the cell's transition to the M phase is the Activation of a specific protein kinase (the cdc protein), which, upon interacting with a cyclin protein, forms a catalytically active complex that phosphorylates numerous cellular proteins required for the execution of mitosis.

The sequence of biochemical reactions triggering mitosis is as follows:

1. Formation of the cdc2 kinase-cyclin complex.

Cdc2 kinase is an enzyme with a molecular mass of 34 kDa; its name is derived from the gene encoding this protein, the cell-division-cycle gene.

Cyclin is a protein with a molecular mass of 45 kDa, the concentration of which gradually increases throughout interphase and drops sharply at the end of mitosis.

2. Phosphorylation of cdc2 kinase complexed with cyclin; phosphorylation occurs at the Tyr-15 and Thr-161 residues of the enzyme. The doubly phosphorylated kinase is inactive but primed to unleash its catalytic activity.

3. Dephosphorylation of the Tyr-15 residue in the cdc2 kinase molecule, yielding a molecular form of the enzyme that is phosphorylated exclusively at Thr-161 and is catalytically active.

This dephosphorylation is catalyzed by a specific protein phosphatase (the cdc25 protein) and serves as the initiating reaction for the entire cascade of biochemical events that drive the cell's transition into the M phase. The activation of cdc25 phosphatase begins upon the completion of DNA SYNTHESIS AND continues throughout the entire period of cellular preparation for mitosis.

4. The catalytically active cdc2 kinase triggers the phosphorylation of cellular proteins involved in initiating the mitotic phase: nuclear membrane lamins, leading to its breakdown; microtubule-associated proteins that form the mitotic spindle; and H1 histones, whose phosphorylation results in the condensation of nuclear chromatin.

It is of considerable interest that the cdc2 kinase engages an enzymatic mechanism that limits its own catalytic activity and, consequently, turns off mitosis.

The restriction of cdc2 kinase activity is achieved through the degradation of its bound cyclin. The "cyclin — monophosphorylated (at Thr-161) cdc2 kinase" molecular complex serves as a substrate for interaction with ubiquitin, a low-molecular-weight (8.5 kDa) protein that acts as a universal Ligand for "tagging" proteins destined for subsequent destruction by proteases. The conjugation of ubiquitin with the cyclin-cdc2 kinase complex leads to the Limited proteolysis of cyclin and the termination of cellular reactions that drive the mitotic process.

The Reactions of the biochemical cycle controlling The entry of a eukaryotic cell into the mitotic phase are illustrated in the diagram (Fig. 22.13).

Fig. 22.13. Scheme of the activation of mitosis initiation proteins by the cdc2 kinase (E) and cyclin (C) complex.