9. DNA & RNA Structure
Nucleotide chemistry, the double helix, alternative conformations, and chromatin organization
Nucleotides: Building Blocks of Nucleic Acids
Nucleotides are the monomeric units of DNA and RNA. Each nucleotide consists of three components: a nitrogenous base, a pentose sugar (deoxyribose in DNA, ribose in RNA), and one or more phosphate groups. A nucleoside lacks the phosphate group and consists of only the base and sugar.
Nomenclature
Nucleoside = nitrogenous base + pentose sugar (e.g., adenosine, guanosine, cytidine, thymidine, uridine)
Nucleotide = nucleoside + phosphate group(s) (e.g., adenosine 5'-monophosphate = AMP)
Purines (bicyclic, 2 rings)
- Adenine (A) — 6-aminopurine, numbering N1–N9
- Guanine (G) — 2-amino-6-oxopurine
Pyrimidines (monocyclic, 1 ring)
- Cytosine (C) — 2-oxo-4-aminopyrimidine, numbering N1–N6
- Thymine (T) — 5-methyluracil (DNA only)
- Uracil (U) — 2,4-dioxopyrimidine (RNA only)
Sugar carbons are designated with primes (C1'–C5') to distinguish them from base atom numbering. The glycosidic bond connects C1' of the sugar to N9 of purines or N1 of pyrimidines. Phosphodiester bonds link the 3'-OH of one nucleotide to the 5'-phosphate of the next, giving nucleic acids a directional backbone running 5' to 3'.
The DNA Double Helix
In 1953, Watson and Crick proposed the double-helical structure of DNA, drawing on X-ray diffraction data from Franklin and Wilkins and the base-composition rules of Chargaff. The model revealed how genetic information is stored and replicated.
Watson-Crick Base Pairing
A=T: Adenine pairs with thymine via 2 hydrogen bonds
G≡C: Guanine pairs with cytosine via 3 hydrogen bonds
A purine always pairs with a pyrimidine, maintaining a constant helix diameter of ~20 Angstroms.
The two strands are antiparallel: one runs 5'→3' while the complementary strand runs 3'→5'. The sugar-phosphate backbones form the exterior, while bases stack in the hydrophobic interior.
Chargaff's Rules
Also: $\frac{[\text{A}] + [\text{G}]}{[\text{T}] + [\text{C}]} = 1$ for double-stranded DNA.
B-DNA Parameters (canonical form)
- Rise per base pair: 3.4 Angstroms
- Base pairs per turn: 10 bp/turn
- Pitch (rise per turn): 34 Angstroms
- Helix diameter: ~20 Angstroms
- Right-handed helix with a major groove (~22 Angstroms wide) and a minor groove (~12 Angstroms wide)
Proteins that recognize specific DNA sequences (e.g., transcription factors) often bind in the major groove, which exposes more base-specific information.
Alternative DNA Structures
DNA can adopt conformations other than the canonical B-form, depending on sequence, ionic conditions, and hydration.
B-DNA
- Right-handed
- 10 bp/turn
- Most common in vivo
- High humidity
A-DNA
- Right-handed
- 11 bp/turn
- Broader, more compact
- RNA-DNA hybrids, dsRNA
Z-DNA
- Left-handed
- 12 bp/turn
- Zigzag backbone
- GC-rich regions, negative supercoiling
DNA Supercoiling
Circular or constrained DNA can be overwound or underwound relative to the relaxed state. The topology is described by the linking number $Lk$, which is a topological invariant for closed circular DNA:
where $Tw$ is the twist (number of helical turns) and $Wr$ is the writhe (number of superhelical turns). The specific linking difference is:
where $Lk_0$ is the linking number of relaxed DNA and $\sigma$ is the superhelical density. Most cellular DNA is negatively supercoiled ($\sigma \approx -0.06$), meaning it is underwound. This facilitates strand separation during replication and transcription.
Topoisomerases
- Type I topoisomerases: Cut one strand, relax supercoils by $\Delta Lk = \pm 1$, no ATP required
- Type II topoisomerases (e.g., DNA gyrase): Cut both strands, pass a duplex through, $\Delta Lk = \pm 2$, require ATP. DNA gyrase introduces negative supercoils in prokaryotes.
RNA Structure
RNA is predominantly single-stranded but forms extensive intramolecular secondary and tertiary structures through self-complementary base pairing. The 2'-OH group on ribose makes RNA chemically more reactive and susceptible to alkaline hydrolysis compared to DNA.
Key Differences: RNA vs DNA
- Sugar: Ribose (2'-OH) vs deoxyribose (2'-H)
- Base: Uracil replaces thymine
- Structure: Mostly single-stranded with hairpins, loops, and bulges
- Stability: Less stable; 2'-OH participates in intramolecular nucleophilic attack on the phosphodiester bond
- Conformation: Adopts A-form geometry in double-stranded regions
Coding and Regulatory RNAs
- mRNA — messenger RNA, encodes proteins
- miRNA — microRNA, post-transcriptional silencing (~22 nt)
- siRNA — small interfering RNA, gene knockdown
- lncRNA — long non-coding RNA, chromatin regulation
Structural and Catalytic RNAs
- tRNA — transfer RNA, amino acid delivery
- rRNA — ribosomal RNA, peptidyl transferase
- snRNA — small nuclear RNA, splicing (U1–U6)
- Ribozymes — catalytic RNAs (e.g., Group I introns)
tRNA Cloverleaf Structure
Transfer RNA (~76 nucleotides) folds into a characteristic cloverleaf secondary structure with four arms:
- Acceptor stem: 3'-CCA terminus, amino acid attachment site
- Anticodon loop: Three-base anticodon for mRNA recognition
- D-loop: Contains dihydrouridine, contributes to tertiary folding
- T$\psi$C loop: Contains pseudouridine ($\psi$), ribosome interaction
The tertiary structure forms an L-shaped molecule with the anticodon and acceptor end at opposite extremities (~75 Angstroms apart).
DNA Stability and Denaturation
The double helix is stabilized by multiple non-covalent interactions. Contrary to a common misconception, base stacking interactions (van der Waals forces and hydrophobic effects between adjacent bases) contribute more to duplex stability than hydrogen bonding between complementary bases.
Forces Stabilizing the Double Helix
- Base stacking: Major stabilizing force. Aromatic ring overlap provides van der Waals attraction and favorable enthalpic contribution.
- Hydrogen bonds: A=T (2 bonds), G≡C (3 bonds). Provide specificity of base pairing more than stability.
- Hydrophobic effect: Exclusion of water from the nonpolar interior of stacked bases.
- Counterion condensation: Cations (Na$^+$, Mg$^{2+}$) neutralize backbone phosphate charges, reducing electrostatic repulsion.
Denaturation (melting) is the separation of the two DNA strands upon heating or exposure to extremes of pH. The midpoint of the transition is the melting temperature $T_m$:
where $N$ is the number of base pairs. Higher GC content and ionic strength increase $T_m$.
Hyperchromicity
Denaturation is monitored by measuring UV absorbance at 260 nm ($A_{260}$). Single-stranded DNA absorbs ~37–40% more UV light than double-stranded DNA (hyperchromic effect). This occurs because base stacking in the duplex quenches UV absorption.
Renaturation (annealing) occurs when denatured DNA is slowly cooled. The kinetics follow second-order behavior and are characterized by $C_0 t$ curves (Britten-Kohne kinetics), where $C_0$ is the initial concentration and $t$ is time. The $C_0 t_{1/2}$ value is proportional to genome complexity.
Nucleic Acid Chemistry
The phosphodiester backbone of nucleic acids can be cleaved enzymatically by nucleases, which are classified by their mode of action and substrate specificity.
Endonucleases
Cleave internal phosphodiester bonds within a nucleic acid strand. Examples: DNase I, RNase A, restriction endonucleases.
Exonucleases
Remove nucleotides from the ends of nucleic acids. Can be 3'→5' or 5'→3' directional. Examples: Exonuclease III, snake venom phosphodiesterase.
Restriction Endonucleases
Bacterial enzymes that recognize specific palindromic sequences (4–8 bp) and cleave both strands. Fundamental tools of molecular biology.
Example:
EcoRI recognizes 5'-G|AATTC-3' / 3'-CTTAA|G-5' → produces sticky ends (4-nt 5' overhangs)
SmaI recognizes 5'-CCC|GGG-3' / 3'-GGG|CCC-5' → produces blunt ends
Chemical synthesis of oligonucleotides proceeds via phosphoramidite chemistry on solid support, adding one nucleotide at a time in the 3'→5' direction. Each cycle involves deprotection, coupling, capping, and oxidation steps. Modern synthesizers achieve coupling efficiencies of >99% per step, enabling synthesis of oligonucleotides up to ~200 nt.
Chromatin Structure
Eukaryotic DNA is packaged with histone proteins into chromatin, achieving a remarkable compaction: ~2 meters of DNA fits within a nucleus ~6 $\mu$m in diameter, a compaction ratio of ~10,000-fold.
The Nucleosome
The fundamental unit of chromatin. A histone octamer consists of two copies each of histones H2A, H2B, H3, and H4 (forming an $(H3 \cdot H4)_2 \cdot (H2A \cdot H2B)_2$ complex). Approximately 147 bp of DNA wraps 1.65 turns around the octamer in a left-handed superhelical path.
Linker DNA (~20–80 bp) connects adjacent nucleosomes. Histone H1 (linker histone) binds at the entry/exit point of DNA on the nucleosome, stabilizing higher-order folding.
Levels of Chromatin Organization
- 2-nm fiber: Bare DNA double helix
- 11-nm fiber: "Beads on a string" — nucleosomes connected by linker DNA
- 30-nm fiber: Compacted arrangement (solenoid or zigzag model), requires H1
- Looped domains: 300-nm fibers, anchored to a protein scaffold
- Metaphase chromosome: Maximum compaction (~1400 nm wide)
Euchromatin
Loosely packed, transcriptionally active. Enriched in acetylated histones (H3K9ac, H3K27ac) and H3K4 methylation.
Heterochromatin
Densely packed, transcriptionally silent. Marked by H3K9 trimethylation and H3K27 trimethylation. Constitutive (centromeres, telomeres) or facultative (inactivated X chromosome).
Histone Modifications (Epigenetic Marks)
Post-translational modifications of histone N-terminal tails regulate chromatin accessibility and gene expression ("histone code"):
- Acetylation (Lys): Neutralizes positive charge, loosens DNA-histone interaction → activation. Writers: HATs; Erasers: HDACs.
- Methylation (Lys, Arg): Context-dependent. H3K4me3 = activation; H3K9me3, H3K27me3 = repression. Writers: HMTs; Erasers: demethylases (KDMs).
- Phosphorylation (Ser, Thr): H3S10ph during mitosis (chromosome condensation), also involved in DNA damage response.
- Ubiquitination (Lys): H2AK119ub (repression via Polycomb), H2BK120ub (transcription elongation).
Key Concepts
- Nucleotides consist of a nitrogenous base (purine or pyrimidine), a pentose sugar, and phosphate groups. Nucleosides lack the phosphate.
- Watson-Crick base pairing: A=T (2 H-bonds), G≡C (3 H-bonds) in antiparallel strands. Chargaff's rules: [A]=[T] and [G]=[C].
- B-DNA is the canonical form: right-handed, 10 bp/turn, 3.4 Angstrom rise, 34 Angstrom pitch. A-DNA and Z-DNA are alternative conformations.
- Supercoiling: Lk = Tw + Wr. Negative supercoiling (underwound) facilitates strand separation. Topoisomerases regulate topology.
- RNA is single-stranded with 2'-OH, uses uracil instead of thymine, and forms extensive secondary structures. Major types: mRNA, tRNA, rRNA, snRNA, miRNA.
- Melting temperature $T_m$ depends on GC content, ionic strength, and sequence length. Denaturation is monitored by the hyperchromic effect at 260 nm.
- Restriction enzymes cleave palindromic sequences, producing blunt or sticky ends. Essential tools for molecular cloning.
- Nucleosomes: 147 bp wrapped around a histone octamer (H2A, H2B, H3, H4). Histone modifications (acetylation, methylation, phosphorylation) regulate chromatin state.