DNA, RNA, and Replication: A Deep Dive into Molecular Biology

DNA and RNA Structure

A nucleoside is formed when a pentose sugar combines with a nitrogenous base (NB). Adding a phosphate group to a nucleoside creates a nucleotide. Nucleotides are linked by O-glycosidic phosphodiester bonds. DNA adopts a right-handed double helix structure, with hydrophobic NBs at the core and hydrophilic pentose and phosphate groups on the exterior. The major groove serves as the interaction site for replication, while the minor groove contributes to structural maintenance. Approximately 10 base pairs span 3.4 nm.

Ribosomal RNA (rRNA)

Ribosomes consist of two subunits: a large subunit (50S in prokaryotes, 60S in eukaryotes) and a small subunit (30S in prokaryotes, 40S in eukaryotes).

DNA Replication

DNA replication must be precise, flexible, reliable, and have repair mechanisms. Key features include:

• Complete replication of the genome
• Coupling to the cell cycle
• Semiconservative replication
• Bidirectional replication from an origin (single origin in eukaryotes, multiple in prokaryotes)
• Simultaneous synthesis
• Semidiscontinuous replication

Requirements for DNA replication include a DNA template strand, deoxynucleotides, Mg⁺², ATP, ribonucleotides, and NAD⁺. Essential enzymes are DNA polymerase III, primase, helicases (prevent reannealing), topoisomerase II (relieves supercoiling), DNA polymerase I, and DNA ligase.

Replication involves a replication fork and mechanisms to prevent reannealing of the separated strands.

DNA Polymerase III vs. I

DNA polymerase III synthesizes DNA in the 5'-3' direction and has 3'-5' exonuclease activity. It is highly processive (1000 nucleotides/sec) and efficient with high fidelity. DNA polymerase I also synthesizes DNA in the 5'-3' direction but has both 3'-5' and 5'-3' exonuclease activity. It is less processive (10 nucleotides/sec) and efficient, primarily functioning in repair.

Steps of replication:

1. Strand separation (initiation, preventing reannealing and torsional stress)
2. Primer synthesis by primase (RNA primer)
3. DNA polymerase III extends the primer
4. Primer removal by DNA polymerase I
5. Gap filling by DNA polymerase I and ligation by DNA ligase (requires AMP, derived from NAD⁺ in prokaryotes and ATP in eukaryotes)

Eukaryotic DNA Replication

• Five DNA polymerases (alpha, delta, epsilon, beta, gamma)
• Primer removal by nucleases
• Telomere replication by telomerase
• Histone assembly and chromatin compaction

DNA Repair

DNA repair mechanisms include direct repair (e.g., photolyase) and excision repair (involving endonucleases).

Transcription

Transcription requires a DNA template strand, ribonucleotides, and Mg⁺². RNA polymerase carries out transcription:

1. Recognizes and binds to specific DNA sequences
2. Unwinds the DNA double helix (no helicase required)
3. Synthesizes RNA in the 5'-3' direction
4. Initiates RNA synthesis without a primer
5. Recognizes termination sites
6. Facilitates RNA folding
7. Is repetitive and processive
8. Lacks exonuclease activity
9. Has a core enzyme and a sigma subunit (for promoter recognition)

Transcription Stages

1. Initiation: Promoter recognition (at -10 and -35 sequences), sigma subunit binds to the promoter, RNA polymerase binds to the DNA template, open complex formation, initiation of RNA synthesis, promoter clearance (sigma subunit is replaced by NusA)
2. Elongation: Formation of a transient RNA-DNA hybrid (10-15 base pairs), nascent RNA synthesis. Hairpin loop formation in the RNA can signal termination and NusA release.
3. Termination: Specific termination sequences or other mechanisms trigger termination.

Eukaryotic Transcription

• DNA is more condensed (acetylation reduces condensation, methylation increases condensation)
• Requires transcription factors (e.g., TATA box in eukaryotes, promoter in prokaryotes)
• RNA processing is essential for maturation (in all eukaryotic RNAs, and tRNA and rRNA in prokaryotes). Processing includes splicing (intron removal), 5' capping, and 3' polyadenylation.

5' capping involves addition of a modified guanine nucleotide to the 5' end of the mRNA. 3' polyadenylation involves addition of a poly(A) tail to the 3' end of the mRNA. Introns are removed by splicing, which recognizes specific sequences at the intron boundaries (GU at the 5' end and AG at the 3' end).