DNA Replication, Transcription, and Translation

Core Review

DNA Structure

DNA is a double helix with a sugar-phosphate backbone and nitrogenous bases (A-T, G-C) connected by hydrogen bonds. The strands are antiparallel: one runs 5'→3', the other 3'→5'. The 5' end has a free phosphate group; the 3' end has a free hydroxyl group. This directionality is critical for replication and transcription.

DNA Replication

DNA replication is semiconservative — each daughter double helix consists of one parental strand and one newly synthesized strand (confirmed by the Meselson-Stahl experiment using ¹⁵N-labeled DNA).

Initiation: Replication begins at origins of replication (multiple in eukaryotes, one in prokaryotic circular DNA). Helicase unwinds the double helix, creating a replication fork. Single-strand binding proteins (SSBs) prevent reannealing. Topoisomerase relieves supercoiling ahead of the fork.

Elongation: DNA polymerase III (in prokaryotes) synthesizes new DNA ONLY in the 5'→3' direction, reading the template strand 3'→5'. Because of this, synthesis on the two template strands differs:

• Leading strand: Synthesized continuously toward the replication fork. Requires only one primer from primase (which synthesizes a short RNA primer to provide a 3'-OH group).
• Lagging strand: Synthesized discontinuously away from the replication fork as short Okazaki fragments (100–200 nucleotides in eukaryotes, 1000–2000 in prokaryotes). Each fragment requires its own RNA primer. DNA polymerase I removes the RNA primers and replaces them with DNA. DNA ligase seals the gaps between fragments.

Key enzymes summary: Helicase (unwinds), primase (RNA primer), DNA polymerase III (elongation), DNA polymerase I (primer removal/replacement), ligase (joins fragments), topoisomerase (relieves supercoiling).

Transcription: DNA → RNA

Transcription is catalyzed by RNA polymerase, which synthesizes RNA in the 5'→3' direction, reading the template strand of DNA 3'→5'. Unlike DNA polymerase, RNA polymerase does NOT require a primer.

Prokaryotic Transcription:

1. RNA polymerase binds to the promoter (specific DNA sequence, including the -10 TATAAT and -35 TTGACA regions in bacteria). Sigma factor helps recognize the promoter.
2. Elongation: RNA polymerase unwinds DNA (~17 bp), adds complementary RNA nucleotides.
3. Termination: either rho-dependent (rho protein binds RNA) or rho-independent (hairpin loop forms in RNA followed by poly-U sequence).

Eukaryotic Transcription: RNA polymerase II transcribes mRNA. Eukaryotes have more complex promoters (TATA box) requiring transcription factors to recruit RNA polymerase.

Post-Transcriptional Modifications (eukaryotes only):

• 5' cap: A modified guanine nucleotide (7-methylguanosine) added to the 5' end. Protects from degradation and aids ribosome binding.
• Poly-A tail: ~200 adenine nucleotides added to the 3' end. Increases stability and aids nuclear export.
• RNA splicing: Introns (non-coding) are removed by the spliceosome (snRNPs), and exons (coding) are joined. Alternative splicing allows one gene to produce multiple protein variants.

Translation: RNA → Protein

Translation occurs on ribosomes, which contain a large and small subunit (80S in eukaryotes). Ribosomes have three tRNA binding sites: A (aminoacyl) site — incoming tRNA, P (peptidyl) site — tRNA holding the growing polypeptide chain, E (exit) site — spent tRNA departs.

The Genetic Code: mRNA is read in triplets called codons. Each codon specifies one amino acid. The code is degenerate (multiple codons for one amino acid), universal (shared across nearly all organisms), and unambiguous (each codon = one amino acid only). Start codon: AUG (methionine). Stop codons: UAA, UAG, UGA.

tRNA Structure: Each tRNA carries a specific amino acid at its 3' end and has an anticodon that base-pairs with the mRNA codon. Aminoacyl-tRNA synthetases "charge" tRNAs by attaching the correct amino acid — this is the step that enforces the genetic code.

Steps of Translation:

1. Initiation: The small ribosomal subunit binds mRNA at the 5' cap (eukaryotes) or Shine-Dalgarno sequence (prokaryotes). The initiator tRNA carrying methionine binds to the start codon (AUG) in the P site. The large subunit joins.
2. Elongation: A new tRNA enters the A site (codon recognition). A peptide bond forms between the amino acid in the P site and the new amino acid in the A site (catalyzed by peptidyl transferase, an rRNA ribozyme). The ribosome translocates (moves one codon down) — the tRNA in the P site moves to E and exits; the tRNA in A moves to P. GTP provides energy. Elongation factors assist.
3. Termination: A release factor recognizes a stop codon in the A site. The polypeptide is released, and the ribosomal subunits dissociate.

Gene Regulation

Prokaryotes — The Lac Operon: The lac operon is an inducible system with three structural genes (lacZ, lacY, lacA) encoding enzymes for lactose metabolism. When lactose is ABSENT, a repressor protein binds the operator, blocking transcription. When lactose is PRESENT, allolactose (an isomer of lactose) binds the repressor, causing it to fall off the operator — transcription proceeds. Catabolite activator protein (CAP) + cAMP enhances transcription when glucose is low (positive control).

Eukaryotes — Multiple Levels of Regulation:

• Promoters: DNA sequences near the transcription start site where general transcription factors and RNA polymerase bind.
• Enhancers: Distant DNA sequences (can be far upstream, downstream, or even within introns) that bind activator proteins and increase transcription rates.
• Transcription factors: Proteins that bind DNA to activate or repress transcription.
• Epigenetics (DOES NOT change DNA sequence):
  • DNA methylation: Addition of methyl groups to cytosine bases (usually at CpG islands) generally silences gene expression.
  • Histone acetylation: Addition of acetyl groups to histone tails neutralizes their positive charge, loosening DNA-histone interaction (euchromatin → active transcription). Deacetylation tightens chromatin (heterochromatin → silenced).

Common Traps

• "DNA polymerase reads 5'→3'": No — DNA polymerase READS the template 3'→5' but SYNTHESIZES the new strand 5'→3'.
• "RNA polymerase needs a primer": False. Only DNA polymerase requires a primer. RNA polymerase can start de novo.
• "All mutations change the protein": Silent mutations (degenerate code) change the codon but not the amino acid. Intron mutations are often neutral.
• "Epigenetic changes alter DNA sequence": They do NOT. Epigenetics changes gene expression through methylation and histone modification.