DNA, Genes and Protein Synthesis

• DNA is a double-stranded polynucleotide forming a double helix
• Each nucleotide: deoxyribose sugar + phosphate group + nitrogenous base
• The two strands run antiparallel (one runs 5' → 3', the other 3' → 5')
• Bases pair by complementary hydrogen bonding: A=T (2 H-bonds), C≡G (3 H-bonds)
• The sugar-phosphate backbone is joined by phosphodiester bonds (covalent)
• The strands are joined by hydrogen bonds between bases

Chargaff's Rules

$\text{Amount of A} = \text{Amount of T}$ $\text{Amount of C} = \text{Amount of G}$ $\frac{A + T}{C + G} \text{ ratio varies between species}$

1. Helicase unwinds the double helix and breaks hydrogen bonds between base pairs
2. Each separated strand acts as a template
3. Free DNA nucleotides line up by complementary base pairing
4. DNA polymerase joins the nucleotides together (forming phosphodiester bonds), working in the 5' → 3' direction
5. One strand is synthesised continuously (leading strand) and the other in fragments (lagging strand — Okazaki fragments)
6. DNA ligase joins the Okazaki fragments together
7. Result: two identical DNA molecules, each with one original and one new strand

This is called semi-conservative replication — each new molecule conserves one original strand.

Evidence: Meselson and Stahl Experiment

• Grew bacteria in heavy nitrogen ($^{15}N$), then transferred to light nitrogen ($^{14}N$)
• After one generation: all DNA was intermediate density (one heavy, one light strand)
• After two generations: 50% intermediate, 50% light
• This confirmed semi-conservative replication

The genetic code has several key features:

Transcription occurs in the nucleus and produces mRNA from a DNA template.

1. RNA polymerase binds to the promoter region of the gene on the DNA
2. The DNA double helix unwinds locally; hydrogen bonds between bases break
3. RNA polymerase moves along the template strand (3' → 5') reading the DNA
4. Free RNA nucleotides align by complementary base pairing:
   • A (DNA) → U (RNA)
   • T (DNA) → A (RNA)
   • C (DNA) → G (RNA)
   • G (DNA) → C (RNA)
5. RNA polymerase joins the nucleotides by phosphodiester bonds, building the mRNA strand in the 5' → 3' direction
6. When RNA polymerase reaches a terminator sequence, transcription stops
7. The pre-mRNA is released

Post-Transcriptional Modification (Eukaryotes)

• Introns (non-coding sequences) are removed by splicing
• Exons (coding sequences) are joined together
• A 5' cap and 3' poly-A tail are added (protect mRNA, aid ribosome binding)
• The mature mRNA then leaves the nucleus through nuclear pores

Translation occurs at ribosomes in the cytoplasm and converts the mRNA code into a polypeptide.

Key Players

• mRNA: Carries the genetic code from nucleus to ribosome
• tRNA (transfer RNA): Small, clover-leaf shaped molecules; each has an anticodon (3 bases complementary to an mRNA codon) and carries a specific amino acid
• Ribosomes: Made of rRNA and protein; have two subunits (large and small); have A site (aminoacyl), P site (peptidyl), and E site (exit)

The Process

1. The small ribosomal subunit binds to the 5' end of the mRNA
2. The start codon (AUG) is recognised; the first tRNA (carrying methionine) binds to the P site
3. The large subunit joins, forming the complete ribosome
4. The next tRNA (with complementary anticodon) enters the A site
5. A peptide bond forms between the amino acids (catalysed by ribosomal RNA — ribozyme activity)
6. The ribosome moves one codon along the mRNA (translocation)
7. The first tRNA exits from the E site, now empty
8. Process repeats: new tRNAs enter the A site, amino acids are added to the growing polypeptide chain
9. When a stop codon (UAA, UAG, or UGA) is reached, a release factor binds
10. The polypeptide is released and folds into its functional 3D shape

Polyribosomes

• Multiple ribosomes can translate the same mRNA simultaneously
• This is called a polyribosome (polysome)
• Allows efficient, rapid protein production

After translation, the polypeptide may be modified:

• Folding into secondary, tertiary, and quaternary structures
• Glycosylation: Addition of sugar groups (in the Golgi apparatus)
• Phosphorylation: Addition of phosphate groups (activates/deactivates proteins)
• Proteolytic cleavage: Removal of sections (e.g., pro-insulin → insulin)

Sickle Cell Anaemia — A Substitution Mutation

• A single base change (A → T) in the haemoglobin gene
• Changes one codon: GAG → GTG
• Amino acid change: glutamic acid → valine (at position 6 of the β-globin chain)
• Valine is hydrophobic, causing haemoglobin molecules to aggregate into fibres
• Red blood cells become sickle-shaped → poor oxygen transport, blocked capillaries
• Heterozygous advantage: Carriers (one normal, one sickle allele) have resistance to malaria

Question: A section of template DNA reads: 3' TAC GGA CTT AAT 5'. Write the mRNA sequence, identify the codons, and determine the amino acid sequence. (4 marks)

Solution:

Template DNA (3'→5'): TAC GGA CTT AAT

mRNA (5'→3'): AUG CCU GAA UUA

Codons: AUG | CCU | GAA | UUA

Amino acids:

• AUG → Methionine (start)
• CCU → Proline
• GAA → Glutamic acid
• UUA → Leucine

Polypeptide: Met – Pro – Glu – Leu

• DNA is a double-stranded antiparallel helix; replication is semi-conservative using helicase, DNA polymerase, and ligase.
• The genetic code is triplet, degenerate, non-overlapping, and universal.
• Transcription (nucleus): DNA → mRNA via RNA polymerase; introns spliced out in eukaryotes.
• Translation (ribosomes): mRNA codons are read; tRNA delivers amino acids; polypeptide is synthesised.
• Mutations (substitution, insertion, deletion) can alter protein structure and function; frameshifts are most severe.