BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 9. NUCLEIC ACIDS

9.4. Structure of nucleic acids

9.4.2. Spatial organization of DNA structure

✵ the sum of pyrimidine nucleotides equals the sum of purine nucleotides (Pur = Pyr);

✵ the number of bases containing amino groups at position 4 of the pyrimidine ring and position 6 of the purine ring (A + C) equals the number of bases with an oxo group at the same positions (G + T);

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Fig. 9.5. Fragments of DNA and RNA polynucleotide chains

✵ the molecular ratio of (G + C) / (A + T) (the coefficient of Specificity) is species-specific. For the DNA of higher animals and plants, as well as certain microorganisms, this ratio is less than 1 (representing the so-called AT-type of DNA), whereas in the DNA of most microorganisms, particularly Bacteria and Fungi, GC pairs predominate (GC-type).

These empirical Chargaff's rules apply exclusively to DNA and are not characteristic of RNA.

These discovered Features of the quantitative relationships among nitrogenous bases indicate that DNA must maintain a rather strict pattern of base pairing—not merely between Purines and Pyrimidines in general, but specifically between adenine and thymine, and guanine and cytosine.

Building on Chargaff's rules and the results of X-Ray Diffraction analyses of Introduction/20.html">DNA Structure, J. Watson and F. Crick proposed an epoch-making double-helix model of the DNA molecule in 1953. There is a poetic rendition of J. Watson's book "The Double Helix," written by J. Field (translated by Oksana Bernyk), highlighting the monumental significance of discovering the three-dimensional structure of DNA.

Listen to the song of how the chiral,

Complex helical structure

Was discovered and unleashed upon the world.

To this structure,

Through rigorous deduction,

Life was given.

From all sides: "Oh! People, what a marvel!" —

The crowd cried out frantically.

While the author took his pen and wrote

A wonderful tale about the little helix:

How a darling from Cambridge

Inadvertently caught the scientific world

HEAD over heels with a leap

In a wonderful moment she transformed.

She came like a dream.

I will tell you the whole truth

How that structure was discovered:

The riddle of the Gene was solved!

Bioprognosis and no mysticism

Better than any fiction.

Amidst the tempest of great thoughts

All hypotheses "came into action":

Base Pairs flickered in the mist

In spite of all obstacles.

That is how it all happened.

With a slight wave of the hand

To connect these bases

H-bonds helped along.

All experiments are overshadowed

Applause rings out in praise!

No matter how you praise it

Still, it's a shame not to know chemistry

Yet, having read—let me tell you—

A single book by Pauling,

He took the models of those bases

And spun them like on a merry-go-round.

He placed them this way, and arranged them that way,

Yet they wouldn't listen at all

Neither thymine nor adenine,

Guanosine ran off somewhere.

He worked tirelessly every day,

And then a letter arrived from Pasadena.

What is going on here? Oh, God,

Pauling's fine model

For atoms, yes, the additional ones,

Which in all exemplary textbooks

As written - only at the very end.

In the guise

(perhaps in a profane phrase)

he gave his feedback on the model

but believe me, he didn't give up!

He started working again

Piecing the bases together somehow.

What more shall I tell you?

Our author tautomers

(Just what is needed) chooses

Nature itself - lends a helping hand.

Two chains (or spirals)

Plus such additional details:

Pauling's bases on the outside,

And phosphates in the middle,

All the facts presented to you are reversed.

Tested via the R-pathway so clear,

To be a hundred percent sincere!

...And so our discovery's tale unfurls,

Of dogmatic nature, brave and bold,

For nations near and far to hold.

It was tough: Rose Cells in grand array

Stood proud for Reflexes that day.

Did Morris call Bragg's book a tablet, pray? —

With wrath and without pity's grace,

Like a kettle boiling over space,

No further words are needed in this case.

What's a fable without a moral core:

Write tablets like these, and explore,

Do not waste precious time in vain —

A fine scientist you shall remain.

And lo, the NOBEL PRIZE draws near —

To bless the world with yet another genius peer!

According to the Watson-Crick model, the DNA molecule consists of two polynucleotide chains forming a right-handed helix around a common axis. The nitrogenous bases are directed toward the interior of the helix, with their planes almost perpendicular to its axis and parallel to one another.

This arrangement results in interplanar base interaction, known as stacking (from the English stack up). This ensures not only favorable Van der Waals contacts between atoms, but also provides additional stabilization due to the overlap of the π-orbitals of the contacting bases' atoms. The stabilization of DNA Double helices is further enhanced by a favorable hydrophobic effect, whereby non-polar bases are shielded from direct contact with the aqueous (hydrophilic) environment. Conversely, the sugar-phosphate backbone, with its polar groups and charged atoms, is exposed, which additionally stabilizes the structure and drives The formation of the Secondary structure of DNA.

The two strands of the DNA molecule have opposite orientations—one is oriented in the 3' → 5' direction, and the other in the 5' → 3' direction. This opposite polarity of the two strands within the helix ensures the correct spatial mutual orientation of the nitrogenous bases.

The polynucleotide chains in the DNA molecule, which are oriented antiparallelly, are linked to each other along their entire length by Hydrogen Bonds. These interchain bonds are formed through the specific interaction of an adenine residue from one strand with a thymine residue from the other strand (two hydrogen bonds), and a guanine residue from one strand with a cytosine residue from the other strand (three hydrogen bonds). The sites of hydrogen bonding between bases in Nucleic Acids are indicated below (Fig. 9.6):

Fig. 9.6. Sites of hydrogen bonding between nitrogenous bases in nucleic acids

The pairing nitrogenous bases are complementary to each other in the sense that hydrogen bonds form between them much more readily than in any other combination. This occurs because the centers of increased and decreased electron density within these bases are optimally aligned relative to one another in these pairs.

In the double-stranded DNA molecule, conformational stability is driven by restricted rotation around the phosphodiester bond and is maintained by the prevailing anti-conformation of the glycosidic bonds and the dominant tautomeric forms of the five nitrogenous bases (see Figs. 9.1 and 9.2)

The DNA helix is regular: one complete turn of the helix (360°) consists of 10 nucleotide pairs, repeating at intervals of 3.4 nm. The distance between adjacent nitrogenous bases along the helical axis is 0.33 nm, and the diameter is 2.37 nm. Each base is rotated by 36° relative to the preceding one.

Fig. 9.7. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF the DNA double helix (B-form)

The spatial arrangement of the strands results in the formation of Major and minor grooves, measuring approximately 2.2 nm and 1.2 nm in width, respectively. Regulatory Proteins associate with the major groove of DNA, specifically interacting with particular atoms of the nitrogenous bases to control Gene Expression without disrupting the complementary base pairing within the double helix structure (Fig. 9.7).

Double-stranded structures in DNA molecules arise not only through the interaction of two complementary polydeoxyribonucleotide strands, but also within a single strand. This occurs when complementary strands contain inverted sequences, known as palindromes (from Greek *palindrome*—running back again)—meaning nucleotide sequences that read the same in both the forward and reverse directions. For example, the sequence

is a palindrome because, when read from 5' to 3', it is identical (GAATTC) on both strands. A distinctive feature of these inverted DNA regions is their ability to form simple or complex hairpin structures under certain conditions (Fig. 9.8).

Fig. 9.8. Example of inverted nucleotide sequences and their ability to form various DNA structures

Virtually all DNA contains palindromic sequences ranging from several to many thousands of base pairs in length. These palindromic structures typically occur in Regions of the DNA molecule that serve as recognition sites for Enzymes and regulatory proteins.

These Structural Features of the double-stranded DNA molecule are characteristic of the B-form, which is dominant under physiological conditions (low salt concentration, high degree of Hydration). However, varying levels of hydration, the presence of cations, and other factors can cause DNA to adopt different Conformations. To date, the following DNA forms have been identified and described: right-handed A-, B-, C-, D-, E-, H-, L-, P-, and the left-handed Z-helix. Nevertheless, only the A-, B-, and Z-forms of DNA have been observed in natural biological systems.

This DNA polymorphism is related to:

✵ the number of base pairs per turn of the double helix;

✵ the distance between the planes of base pairs and the angle they form with the helical axis;

✵ the diameter of the helix;

✵ the handedness (right or left) of the double helix, which depends on The nucleotide sequence, the degree and direction of supercoiling, chemical modifications of the nitrogenous bases, and the concentration of solutes in the solution, particularly Metal Ions and Polyamines.

Selected geometric data for DNA isoforms are presented in Table 9.3.

Table 9.3

Characteristics of Selected DNA isoforms

Changes in DNA conformation depending on environmental conditions occur because the Bond Angles between bases and pentoses are adaptable, and the pentose-phosphate backbone is sufficiently flexible to accommodate alternative double-Helical structures. Notably, some of these forms can transition into one another when specific parameters are altered or certain factors are applied. Within The Cell, DNA typically exists in the B-form, though specific regions may adopt A-, Z-, or other conformations. Importantly, the A-form of DNA closely resembles the structure characteristic of double-stranded RNA or DNA-RNA hybrid molecules. For clarity, several alternative conformations are illustrated schematically (Fig. 9.9).

Fig. 9.9. DNA isoforms

Under certain conditions, DNA regions characterized by alternating purine and pyrimidine nucleotides (such as GC or AT repeats) adopt a left-handed helical conformation (Z-form). In this state, the distance between adjacent base pairs increases to 0.371 nm (see Table 9.3), and the number of base pairs per turn rises to 12. The DNA backbone appears zig-zagged because the alternating guanine-cytosine nucleotide sequences and deoxyribose residues undergo conformational changes, causing the line connecting the phosphate groups to bend every two base pairs.

Factors stabilizing the Z-form of DNA include: 1) binding of Z-DNA-specific proteins; 2) methylation of the carbon atom at the 5-position of certain deoxycytidine residues; and 3) the presence of specific cations, such as spermine and spermidine. It is hypothesized that Z-DNA may play a role in regulating the expression of genes located both in close proximity and at significant distances from the Z-region.

Cellular linear DNA takes the form of an elongated molecule packed into a compact structure, forming coiled and supercoiled configurations. This indicates that the double-stranded DNA molecule can undergo further spatial folding into a specific tertiary structure—a supercoil or a circular form. Bacterial Plasmids, the Chromosomes of certain bacteria, most Mitochondrial and Chloroplast DNAs, and animal DNA-containing Viruses are represented by covalently closed circular DNA molecules. These closed circles can exist in either a relaxed or a supercoiled state (Fig. 9.10).

Fig. 9.10. Linear, relaxed cyclic, and supercoiled forms of DNA

Converting a relaxed DNA structure into a supercoiled one requires a certain expenditure of energy, resulting in a more compact molecule that can be packaged into a small volume. Supercoils formed by counter-clockwise coiling—opposite to the right-handed twist of the DNA double helix—are termed negative. When a DNA molecule transitions to another type of supramolecular structure, energy can decrease due to the formation of negatively supercoiled regions. An example of such a transition is the Separation of DNA strands during template-directed processes, such as DNA Synthesis (Replication) or RNA Synthesis (METABOLISM/31.html">Transcription). Enzymes that catalyze topological Changes in the DNA molecule are called topoisomerases.

The supercoiled conformation of DNA is characteristic of the prokaryotic nucleoid and the chromosomes of higher organisms. Such a tertiary structure is stabilized by electrostatic forces, where negatively charged phosphate groups are partially neutralized by positively charged metal ions and polyamines, or by basic amino acid residues of proteins. As a result of these interactions, DNA undergoes Condensation, reducing its volume by a factor of thousands. For example, the DNA molecule of one of the smallest Human chromosomes is about 3 cm long, whereas the total length of all DNA in a single cell is about 2 m, yet it is packed within a nucleus with a diameter of only about 5 µm.

Each DNA molecule is packaged into a separate chromosome, which contains an amorphous nucleoprotein complex called Chromatin; the state of this complex varies depending on the Cell Cycle. In the resting phase, chromatin is evenly distributed throughout the nuclear volume. During Cell Division, chromatin forms compact bodies known as chromosomes. Morphologically, a distinction is made between euchromatin and heterochromatin, the latter being more condensed than euchromatin, which corresponds to regions of chromosomes undergoing active transcription.

Chromatin is a supramolecular structure composed of DNA, Histones, and other Nuclear Proteins (non-histone proteins). Approximately 2/3 of chromatin mass consists of proteins and 1/3 of DNA; chromatin also contains RNA (up to 10%), a minor percentage of Lipids, and certain metal ions (Mg²⁺, Ca²⁺, Fe²⁺). The term "histones" refers to several groups of related basic proteins with a Molecular Weight of 11-21 kDa. A characteristic feature of histones is their high Lysine and/or Arginine content. Due to their positive charge, histones form ionic bonds with the negatively charged phosphate groups located on the outer surface of the DNA double helix.

There are five MAIN TYPES OF histones: H1, H2A, H2B, H3, and H4. Two molecules of each of the H2A, H2B, H3, and H4 histones form an octamer (the nucleosome core) (Fig. 9.11, A), which is wrapped with a 146-base-pair DNA segment (1.75 turns). Such a complex of histone proteins and DNA is the fundamental structural unit of chromatin, called a nucleosome. The assembly of nucleosomes involves nucleoplasmin, an anionic nuclear protein capable of reversibly binding to the histone octamer, thereby blocking the ability of histones to interact non-specifically with negatively charged structures such as DNA. Nucleoplasmin exhibits selectivity for specific DNA regions and presumably creates a specific ionic environment within The Nucleus that facilitates Histone-DNA Interaction and nucleosome assembly.

The DNA region located between adjacent nucleosomal particles is called linker DNA and consists on average of 60 base pairs. The H1 histone molecule is not part of the nucleosome core and does not participate in wrapping DNA around the histone octamer. Instead, histone H1 contacts DNA where the double helix enters and exits the nucleosome core, essentially "stitching" the DNA at the points where it begins and ceases to wrap around the core (Fig. 9.11, B).

Further folding of nucleosomes in the nucleus presumably depends on the interaction of H1 histones with the regions of double-stranded DNA connecting the nucleosomes. The topology of this interaction, which leads to the formation of internucleosomal connections, is not yet fully understood. However, Electron Microscopy of the interphase nucleus has revealed a solenoid-like structure with a diameter of 30 nm, commonly referred to as the 30 nm fiber.

Fig. 9.11. Model of the nucleosome core structure (A) and nucleosomes (B)

It is hypothesized that in this packing arrangement, disc-shaped nucleosomes with a diameter of 10 nm and a height of 5 nm Touch each other at their edges, orienting their flat surfaces along the axis of the fiber. These fibers presumably also coil with a pitch of 6 nucleosomes per turn, resulting in the formation of a 30 nm solenoid (Fig. 9.12).

When chromatin condenses to form a metaphase chromosome, the solenoid structures form loop-like domains 300 nm in diameter that are attached to a protein scaffold (the nuclear scaffold). The nuclear scaffold consists of chromosomal structural proteins upon which the final condensation of chromatin takes place.

Fig. 9.12. Model of the 30 nm chromatin fiber representing a supranucleosomal level of DNA compaction

Subsequent compaction of DNA leads to the formation of additional superstructures. Approximately 20 loops form so-called minidisks, which in turn stack into columns (700 nm) (Fig. 9.13).

The consequence of this DNA packaging is the formation of condensed chromatin, typical of eukaryotic metaphase chromosomes, which shortens the DNA molecule by nearly 8,000- to 10,000-fold.

Certain amino acid residues (arginine, lysine) and terminal amino groups of histones within nucleosomes can undergo chemical modification: phosphorylation, methylation, Acetylation, or ubiquitination, among others. Such covalent modifications can alter the charge and conformation of histones, which in turn affects the interactions among histones themselves and with DNA. These chemical modifications enable conformational rearrangements of chromatin, which are crucial for the Regulation of Gene Expression.

Fig. 9.13. Scheme of DNA compaction in chromosomes

Non-histone chromatin proteins. Eukaryotic Cell nuclei contain a diverse array of DNA-binding non-histone proteins. These include:

✵ structural proteins that participate in forming the supranucleosomal levels of chromosome folding;

✵ a large group of enzymes that drive replication, transcription, repair, and the chemical modification of chromatin components;

✵ the most functionally diverse group of regulatory proteins, which control The activity of the aforementioned enzymes as well as the accessibility of specific DNA regions to these enzymes. Examples include the family of site-specific proteins such as zinc fingers, leucine zippers, and homodimers. They possess a specialized structure and belong to the so-called transcription factors that bind to regulatory regions of genes, thereby influencing expression. The structural-regulatory proteins permanently associated with chromatin include high mobility group proteins (HMG proteins). These proteins are characterized by relatively small sizes (up to 30 kDa) and a high content of charged amino acid residues. They exhibit an affinity for and bind specifically to nucleosomes located in transcriptionally active regions of chromatin.