Nucleotides, DNA, and RNA

The genetic information of an organism is stored in the form of nucleic acids. Nucleic acids, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are long linear polymers composed of nucleotide building blocks. Each nucleotide is comprised of a sugar, a phosphate residue, and a nitrogenous bases (a purine or pyrimidine). DNA is longer than RNA and contains the entire genetic information of an organism encoded in the sequences of the bases. In contrast, RNA only contains a portion of the information and can have completely different functions in the cell.

DNA is structurally characterized by its double helix: two opposite, complementary, nucleic acids strands that spiral around one another. The DNA backbone, with alternatively linked sugar and phosphate residues, is located on the outside. The bases are located inside the helix and form the base pairs adenine and thymine or guanine and cytosine, which are linked by hydrogen bonds.

The human genome comprises 3.2 x 10⁹ base pairs, which are distributed over 23 pairs of chromosomes. Each chromosome is a linear DNA molecule of a certain length. The chromosome is only well visualized under the light microscope during the metaphase of mitosis, as it is maximally condensed during this phase. Chromosomes are present as pairs in most cells of the body. One chromosome in each of the 23 pairs originates from the mother and the other from the father.

Both interrelated chromosomes are termed homologous because they each have a variant of the same gene. Alterations in the number or structure of the chromosomes lead to various conditions, e.g., developmental disorders. Chromosomal assessment with different molecular biology and cytogenetic methods often allows for a clear diagnosis.

Nucleotides

• Structure
  • Nitrogenous base (a purine or pyrimidine)
  • Pentose sugar
  • Phosphate group
• Bonds
  • Nucleoside: base and sugar (ribose or deoxyribose), linked by an N-glycosidic bond
  • Nucleotide: nucleoside and phosphate group, linked by a 3'-5' phosphodiester bond

NucleoSides consist of base and Sugar (deoxyribose). NucleoTides consist of base, deoxyribose and phosphaTe.

Nucleobases

• Cytosine has 3 H-bond donors and forms a strong bond with guanine, which has 3 H-bond acceptors.
  • More stable than bonds consisting of 2 H bonds (A-T)
  • The higher the number of cytosine-guanine bonds in DNA, the higher its melting temperature.
• Other than uracil, there are many other bases that may be created after the initial nucleic acid chain formation, for example:
  • Hypoxanthine
    • Created from adenine via deamination during RNA editing
    • Present as inosine in tRNA and plays an important role in ensuring proper wobble base pair translation (see “Wobble hypothesis”)
  • Xanthine
    • Intermediate of purine metabolism
    • Created from guanine via deamination
• Amino acids required for purine synthesis
  • Glycine
  • Aspartate
  • Glutamine
• See “Purines and pyrimidines” for more details.

“A mean person GAGs a PURring cat!” (Three Amino acids, Glycine, Aspartate, and Glutamine, are necessary for PURine synthesis.)

“C-G stabilizes DNA Crazy Good!” (C-G bonds are extremely stable.)

“PYRates Capture 1 Undersea Treasure.” (PYRimidine bases: Cytosine, Thymine, and Uracil and consist of 1 ring.)
“PURe A Glass for 2.” (PURine bases are Adenine and Guanine and consist of 2 rings.)

Thymine contains a methyl group and is only found in DNA; uracil is only found in RNA.

Nucleic acid sugars

• Structure: The sugar found in nucleic acids is a pentose, which has a five-atom ring.
  • DNA is deoxyribose
  • RNA is ribose
• Pentose binds
  • Bases via N-glycosidic bonds
  • Phosphate residue via phosphodiester bonds

Phosphate group

• A nucleotide can have one, two, or three phosphate groups (also termed “nucleoside monophosphate”, “diphosphate”, and “triphosphate”, respectively).
• Nucleic acids are composed of nucleoside monophosphates.
• Nucleoside diphosphates and nucleoside triphosphates (e.g., ATP) are found in biochemical processes requiring energy
  • The phosphoanhydride bonds store a high amount of energy that can be utilized in biochemical processes when targeted by 3' hydroxyl attack.
  • The nucleotide that is added to the 5' end of the nucleic acid initially has three phosphate groups. The splitting of the two end phosphate groups supplies the energy necessary for the phosphodiester bonds that build the DNA backbone.

Nucleotide and nucleotide derivatives have important functions in the body.

• Building blocks of nucleic acids
• Source of energy: : especially as a universal energy carrier of the cell in the form of ATP, but also GTP
• Signal molecules: especially the second messenger cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) , both phosphoric esters
• Activators for the transfer of groups: Through the potential of forming energy-rich bonds, nucleotides are able to transfer a molecule onto another in biosynthesis, e.g.:
  • UDP-Glucose is an active form of glucose in glycogenesis.
  • Dietary choline can be activated to citicoline by CTP and be used in the synthesis of phosphatidylcholine.
  • 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in sulfatide synthesis.
  • S-Adenosyl methionine (SAM) is formed from methionine and serves as a cofactor in methylation reactions.
• Regulators: enzyme reactions in signal transduction pathways (e.g., activates GTP G proteins)
• Carrier molecules: e.g., the electron carrier nicotinamide adenine dinucleotide (NAD⁺) and flavin adenine dinucleotide (FAD) as a component of coenzymes in redox reactions

The energy carrier ATP contains ribose and not deoxyribose as a sugar, and therefore has a 2' OH group.

Nucleic acids

• Long, linear chains (polymers) of nucleotides
• Alternating sugar and phosphate residues of individual nucleotides, linked by phosphodiester bonds, form the backbone
• Primary structure of nucleic acids: nucleotide sequence in the chain
• Phosphodiester bonds are negatively charged.
  • Negative charges stabilize the nucleic acids.
  • Phosphodiester bonds cannot be easily hydrolyzed like other esters.
• The chemical composition of nucleic acids (DNA and RNA) and their structure of repetitive nucleotide units allow them to function as both information carrier and mediator.

Comparison of DNA and RNA

Overview of double-stranded DNA

Organization of the human genome

• Double-stranded chain of deoxyribonucleotides in cells
• Both strands are complementary to each other and run anti-parallel.
  • Nucleotides form single-stranded DNA that stabilizes into double-stranded DNA
  • DNA conforms into right-handed double helix that binds histone octamers to form nucleosomes (appear as “beads on a string” under electron microscopy)
  • Chromatin formation begins, which is then further compacted
  • During replication (mitosis or meiosis), chromatin maximally condenses into chromosomes (only visible during metaphase under light microscopy)

Double helix

• 3D structure of DNA in which two polynucleotide strands are intertwined, stabilized by:
  • Specific base pairing via hydrogen bonds (H bonds) between complementary nucleobases of DNA
    • A-T bonds consist of 2 H bonds
    • G-C bonds consist of 3 H bonds, resulting in a stronger bond (an ↑ in G and C in DNA → ↑ melting temperature of DNA)
  • Hydrophobic effect: The negatively charged sugar-phosphate backbone is located on the outside of the helix, the bases on the inside.
  • Base stacking: The base pairs are stacked on one another (stacking interactions) and interact through van der Waals forces, which have an additional stabilizing effect.
• Double helix has a minor groove and a major groove.

Conformations

• B conformation (B-DNA)
  • Most prevalent
  • Right-handed double helix
  • 10 base pairs per helical twist to a length of 3.4 nm
  • Diameter of the helix: 2 nm
  • Bases are approx. perpendicular to the helix axis.
• A conformation (A-DNA)
  • Right-handed double helix, although broader and shorter than B-DNA
  • Base pairs are not perpendicular to the helix axis but are slightly inclined toward the axis.
  • Dehydrated form, i.e., present under experimental conditions and not in vivo.
• Z conformation (Z-DNA)
  • Left-handed double helix
  • Stretched longer than B-DNA resulting in a smaller diameter
  • Occurs in GC-rich sequences, although they are generally rare under physiological conditions
  • The phosphate groups of the DNA backbone form a zigzag pattern.

Base pairs in DNA: guanine pairs with cytosine (3 H bonds), adenine pairs with thymine (2 H bonds).

Supercoils

• Description: winded double helix , also termed “superhelix”
• Occurrence: especially in circular DNA molecules
  • In prokaryotes: chromosome of bacteria, plasmids
  • In eukaryotes
    • Mitochondrial DNA (circular)
    • “Inflexible” segment of linear, chromosomal DNA
• Function: Supercoiled DNA molecules have a more compact structure than the relaxed form of DNA.

Palindrome

• Description
  • A palindrome is a sequence that reads the same forwards and backward (e.g., eye, level, madam).
  • The molecular biological use of the term “palindrome” is for inverted repeats (repeated sequence in the opposite direction).
• Occurrence
  • In palindromic sequences, a sequence of base pairs occurring over a certain segment is read identically on both complementary DNA strands, i.e., the sequence always reads the same on both strands in a 5'→3' direction.
  • Bases may be present between the palindromic sequences that are not complementary.
  • These segments are self-complementary and can form a hairpin loop.
  • Results in the formation of a cross-shaped structure in double-stranded DNA
• Function: Some proteins that are capable of binding DNA require palindromic sequences as a recognition sequence, e.g., steroid hormone receptors or restriction enzymes.

Chromatin

• Definition: complex of DNA and its associated proteins (both histones and non-histones) structured as repetitive units (nucleosomes)
• Functions
  • Condensation and organization of DNA (a very large molecule) allow for storage inside the nucleus and are important for gene regulation
  • Chromatin remodeling
    • Opening chromatin structure from a compact state to a more accessible arrangement
    • Allows for transcription factors and RNA polymerase to access specific loci of genes
    • Facilitated by various enzyme remodelers (e.g., SWI/SNF ATPases), histone post-translational modifications (see below), and direct modification of DNA itself (e.g., DNA demethylation).
• Types
  • Heterochromatin
    • Contains inactive DNA because the highly condensed, steric conformation does not allow transcription
    • Darker on electron microscopy (EM)
    • DNA is highly methylated and deacetylated
  • Euchromatin
    • Contains active DNA because the less condensed steric conformation makes DNA accessible for transcription
    • Lighter on EM

Heterochromatin is Hooked tight while Euchromatin is Easygoing.

Histones

• Definition: group of proteins that bind to DNA in the nucleus of eukaryotes to support the structure of chromatin
• Characteristics
  • Positively charged through the high percentage (∼ 25%) of basic amino acids (arginine and lysine)
  • Strong ionic interactions with negatively charged DNA (through phosphate groups on DNA)
  • Synthesized during S phase in the cytosol and transported to the nucleus
• Types: There are four core histones and a linker.
  • 4 Core histones: H2A, H2B, H3, H4
    • 2 molecules of each core histone form the nucleosome 8-protein complex core, a histone octamer, around which the DNA is wound in segments
    • Controls gene expression via reversible post-translational modification of histones (acetylation, methylation, phosphorylation, ubiquitinylation, Sumoylation, ADP-ribosylation)
      • Histone methylation
        • Occurs via histone methyltransferase, which targets lysine or arginine residues
        • Methylation usually suppresses transcription by enabling tighter DNA coiling.
        • Depending on the location and number of methyl groups that are attached, methylation can also promote transcription.
      • Histone acetylation
        • Acetylation of specific lysine residues (positively charged) in histone proteins → less positively charged histones → weaker binding of DNA → relaxation of DNA coiling → ↑ transcription activity
        • Similarly, histone deacetylation via histone deacetylase tightens the coiling of DNA and decreases transcription activity (see “Histone modification”).
        • Clinical implications: pathogenesis of Huntington disease (dysregulated acetylation, e.g., histone deacetylation altering gene expression); thyroid hormone-induced acetylation that influences thyroid hormone synthesis
  • Linker histone (H1)
    • Structure: not completely known, but a different, less uniform structure than core histones
    • Function: binds to linker DNA and to the nucleosome, leading to stabilization of the chromatin fiber.

Histone Methylation Mutes transcription. Histone Acetylation Activates transcription.

Nucleosome (nucleosome core particle)

• Definition: a structural and functional complex of DNA (∼ 150 bp) and histone octamer that gives chromatin its “beads on a string” appearance
• Structure
  • DNA wraps around the nucleosome core with ∼ 1.8 twists
  • Nucleosomes are linked to one another through linker DNA (short DNA segment of variable lengths)
• 30 nm chromatin fiber (solenoid)
  • Nucleosome strand that is spirally bound to fibers with a diameter of 30 nm
  • Each twist of the 30 nm fiber contains ∼ 6 nucleosomes.
• Chromatin loop
  • Condensed form of DNA beyond the nucleosome and 30 nm fibers
  • The histone H1 and nonhistones are involved in the formation of loops.

Chromosomes

See “Basics of human genetics” for more information.

• Description
  • A denser packaging of chromatin that only becomes visible under the microscope during cell division (especially in metaphase)
  • Number of chromosomes in the human genome:
    • Somatic cells contain 23 pairs of homologous duplicated chromosome pairs (46 chromosomes in total).
    • Germ cells only contain 23 single-stranded, unduplicated chromosomes.
• Structure: A chromosome pair consists of 2 identical chromatids connected at the center by a centromere.

Human genome

• The human genome consists of ∼ 3.2 billion base pairs (bp).
• The DNA stored in a human cell would total ∼ 1.8 m in length.
• In addition to the nuclear genome (found in the nucleus), there is also a mitochondrial genome that largely codes for RNA-associated proteins

Nuclear genome

• ∼ 10% contains genes and related sequences
  • ∼ 3% of the nuclear genome codes for proteins (including introns) and RNAs.
  • ∼ 1% of the nuclear genome only codes for exons.
• ∼ 90% does not contain genes
  • The function of ∼ 50% of DNA sequences is unknown.
  • ∼ 45% is composed of repetitive sequences (repetitive genetic elements).
    • Simple repetitive DNA elements (tandem repeats)
      • Satellite DNA: repetitive sequences of up to 18,000 nucleotides
      • Minisatellite DNA: repetitive sequences from 3–100 nucleotides
      • Microsatellite DNA: repetitive sequences from 2–6 nucleotides
    • Previously mobile genetic elements (such as transposons, LTR , non-LTR, LINE , SINE )
  • ∼ 24% of the genome is spanned by introns.

Mitochondrial genome (mitochondrial DNA, mtDNA)

• Circular genome of ∼ 16,500 bp
• Mitochondrial DNA does not utilize histones for DNA packaging.
• Over 90% of mtDNA codes for structural genes, including for mRNA, tRNA, and rRNA.
  • 13 genes that code for proteins, i.e., for mRNAs
  • 22 genes for tRNAs
  • 2 genes for rRNA

RNA classes and their structure

RNAs can be differentiated into various types, which differ in their length, structure, and function. Depending on the type, RNA can be a single-stranded or double-stranded segment.

To remember the features of the two tRNA arms, think: “Dihydrouridine and Detection” for the D-arm and “tRNA Tethering” for the T-arm.

“CCA Can Catch Amino acids” (function of the 5'-CCA-3' sequence in tRNA).