Learning Objectives
- Understand the structure of DNA and RNA
- Learn about DNA replication, transcription, and translation
- Study the genetic code and gene regulation
- Understand the Human Genome Project and DNA fingerprinting
Key Concepts
DNA Structure
Watson and Crick Model (1953): Double helix with two antiparallel polynucleotide chains. Sugar-phosphate backbone on outside, nitrogenous bases inside. Base pairing: A=T (2 H-bonds), G≡C (3 H-bonds) — Chargaff's rules. Pitch of helix: 3.4 nm (34 Angstroms). Distance between base pairs: 0.34 nm. ~10 bp per turn. Right-handed helix (B-DNA). X-ray crystallography data by Wilkins and Franklin was crucial for the model.
Nucleosome model of chromatin: DNA wraps around histone octamer (2 each of H2A, H2B, H3, H4) ~1.65 times (~200 bp). H1 histone — linker. Euchromatin: Loosely packed, transcriptionally active. Heterochromatin: Tightly packed, transcriptionally inactive.
DNA Replication
Semi-conservative replication — proved by Meselson and Stahl experiment using E. coli and N15/N14 (density gradient centrifugation). Each new DNA has one old and one new strand. Enzyme: DNA polymerase III (main replicating enzyme, adds nucleotides 5'→3'). Helicase: Unwinds DNA. SSB proteins: Stabilize single strands. Primase: Synthesizes RNA primer. Leading strand: Continuous synthesis (5'→3'). Lagging strand: Discontinuous synthesis — Okazaki fragments joined by DNA ligase. Origin of replication (ori): Specific sequence where replication starts. Eukaryotes have multiple origins.
Transcription (DNA → RNA)
RNA polymerase reads template strand (3'→5') and synthesizes mRNA (5'→3'). In prokaryotes: single RNA polymerase. In eukaryotes: RNA Pol I (rRNA), RNA Pol II (mRNA — hnRNA), RNA Pol III (tRNA, 5S rRNA, snRNA). Promoter: Upstream sequence where RNA polymerase binds (TATA box at -25 in eukaryotes). Terminator: Sequence signalling end of transcription.
Post-transcriptional modifications (eukaryotes): hnRNA → mRNA by: (1) Capping (methyl guanosine cap at 5' end), (2) Tailing/Polyadenylation (poly-A tail at 3' end), (3) Splicing (removal of introns, joining of exons). Split genes: Eukaryotic genes have exons (expressed) and introns (intervening, non-coding).
Translation (mRNA → Protein)
Occurs on ribosomes. Genetic code: Triplet code — 64 codons for 20 amino acids. AUG = start codon (codes for methionine). UAA, UAG, UGA = stop codons (nonsense codons). Code is universal, degenerate (multiple codons for same amino acid), non-ambiguous, non-overlapping, and comma-less.
tRNA: Adapter molecule with anticodon (reads codon on mRNA) and amino acid attachment site. Clover-leaf shaped. Translation steps: (1) Initiation — small ribosomal subunit + mRNA + initiator tRNA (Met-tRNA) at AUG. (2) Elongation — amino acids added by peptide bond formation (peptidyl transferase). (3) Termination — stop codon reached, release factors bind, polypeptide released.
Gene Regulation — Lac Operon
Proposed by Jacob and Monod in E. coli. Structural genes: lacZ (beta-galactosidase), lacY (permease), lacA (transacetylase). Repressor (coded by i gene) binds operator in absence of lactose → genes OFF. When lactose (inducer) present → binds repressor → repressor cannot bind operator → genes ON (inducible system). Lactose is converted to allolactose (actual inducer).
Human Genome Project (HGP)
Launched in 1990, completed in 2003. Goals: sequence all 3.2 billion base pairs, identify all genes (~20,000-25,000). Key findings: less than 2% DNA codes for proteins; repetitive sequences make up large portion; average gene size ~3000 bp. Methodologies: BAC/YAC cloning, DNA sequencing (Sanger method). SNPs (Single Nucleotide Polymorphisms) — ~1.4 million identified.
DNA Fingerprinting
Developed by Alec Jeffreys. Based on VNTR (Variable Number Tandem Repeats) — also called minisatellites. VNTRs are highly polymorphic. Process: DNA isolation → restriction enzyme digestion → electrophoresis → Southern blotting → hybridization with VNTR probe → autoradiography. Applications: forensics, paternity disputes, evolutionary studies.
Summary
DNA is the genetic material with a double helix structure. Replication is semi-conservative. Transcription converts DNA to RNA; translation converts mRNA to protein. The genetic code is universal and degenerate. Gene expression is regulated by operons in prokaryotes. The Human Genome Project mapped the entire human genome. DNA fingerprinting uses VNTRs for identification.
Important Terms
- Semi-conservative replication: Each daughter DNA has one parent and one new strand
- Okazaki fragments: Short DNA segments on lagging strand
- Genetic code: Triplet codons specifying amino acids; universal and degenerate
- Operon: Cluster of genes regulated together in prokaryotes
- Introns: Non-coding sequences removed during RNA splicing
- Exons: Coding sequences that are expressed in final mRNA
- VNTR: Variable Number Tandem Repeats used in DNA fingerprinting
- Central dogma: DNA → RNA → Protein (proposed by Crick)
Quick Revision
- DNA: A=T (2H), G≡C (3H); pitch 3.4 nm, 10 bp/turn
- Semi-conservative replication: Meselson & Stahl experiment
- RNA Pol II makes mRNA (hnRNA) in eukaryotes
- hnRNA processing: capping + tailing + splicing → mature mRNA
- AUG = start (Met); UAA, UAG, UGA = stop codons
- Lac operon: lactose (inducer) → repressor inactive → genes ON
- HGP: ~20,000-25,000 genes; less than 2% coding DNA
- DNA fingerprinting: VNTR polymorphism; developed by Alec Jeffreys