Molecular Basis of Inheritance

The central dogma: DNA → RNA → Protein

DNA → transcription → mRNA → translation → protein. DNA also copies itself by replication.

Structure of DNA

Building block: the nucleotide

• Nucleotide = nitrogen base + sugar + phosphate.
• Nucleoside = nitrogen base + sugar (no phosphate).
• Purines (2 rings): Adenine, Guanine.
• Pyrimidines (1 ring): Cytosine, Thymine (in DNA), Uracil (in RNA instead of T).

The double helix (Watson and Crick, 1953)

• DNA is a twisted ladder: the sides are sugar–phosphate and the steps are base pairs.
• The two strands run in opposite directions (antiparallel): one 5'→3', the other 3'→5'.
• A = T (2 hydrogen bonds) and G ≡ C (3 hydrogen bonds).
• Pitch (one full turn) = 3.4 nm, with 10 base pairs per turn, so the distance between two base pairs = 0.34 nm.
• Chargaff's rule: in DNA, A = T and G = C, so purines = pyrimidines.
• Watson and Crick built their model using X-ray pictures by Maurice Wilkins and Rosalind Franklin.

How 2 metres of DNA fits in a nucleus: packaging

DNA double helix → wraps around a histone octamer (8 histones) → nucleosome (~200 bp) → "beads on a string" → chromatin fibre → chromosome (seen during cell division).

• Histones are positively charged (rich in lysine and arginine), so they hold negatively charged DNA like a magnet.
• Euchromatin: loosely packed and active (genes are read).
• Heterochromatin: tightly packed and inactive.

Proving DNA is the genetic material

• Viruses with radioactive DNA (³²P) infected bacteria → radioactivity was found inside the bacteria.
• Viruses with radioactive protein (³⁵S) infected bacteria → radioactivity stayed outside the bacteria.
• Conclusion: DNA, not protein, enters the bacteria and carries the genetic information.

Why DNA is better than RNA as genetic material

• RNA has an extra 2'-OH group, which makes it reactive and unstable.
• RNA mutates faster.
• DNA is double-stranded and stable. That's why DNA stores information, while RNA mostly carries messages.
• RNA was probably the first genetic material ("RNA world"), and some RNAs work as enzymes (ribozymes).

DNA replication: making a copy

• DNA copies itself in a semi-conservative way: each new DNA has 1 old strand + 1 new strand.
• Proved by Meselson and Stahl (1958) using heavy nitrogen (¹⁵N) and normal nitrogen (¹⁴N) in E. coli.

1. Start: all DNA has heavy ¹⁵N (band at the bottom of the tube).
2. After 1 generation in ¹⁴N: all DNA is hybrid ¹⁵N/¹⁴N (one middle band).
3. After 2 generations in ¹⁴N: half is hybrid and half is light ¹⁴N (a middle band and a top band).

The replication team (enzymes)

• Leading strand: made continuously.
• Lagging strand: made in short pieces called Okazaki fragments, which ligase joins.
• Replication starts at a point called the origin of replication (ori), and happens in the S phase of the cell cycle.

Transcription: DNA → RNA

• Only one strand of DNA is used as a template.
  • Template strand (3'→5'): read to make RNA.
  • Coding strand (5'→3'): same sequence as the RNA, with T in place of U.
• A transcription unit has 3 parts: promoter (start signal) → structural gene → terminator (stop signal).

RNA processing in eukaryotes

The first copy (hnRNA) is edited before it leaves the nucleus:

hnRNA (exons + introns) → splicing (remove introns, join exons) → capping (add a methyl guanosine cap at the 5' end) → tailing (add a poly-A tail at the 3' end) → mRNA.

The genetic code

• Worked out by George Gamow, Har Gobind Khorana and Marshall Nirenberg.
• Severo Ochoa's enzyme (polynucleotide phosphorylase) helped make RNA for the experiments.

Translation: RNA → protein

• tRNA is the "adapter". One end has the anticodon (it reads the mRNA codon); the other end carries the matching amino acid. Its 2D shape is a clover leaf and its 3D shape is an inverted L.
• Ribosome is the protein factory. In bacteria, its 23S rRNA works as an enzyme (a ribozyme) that makes the peptide bonds.

1. Charging: each tRNA picks up its amino acid (uses ATP).
2. Initiation: the ribosome binds the mRNA at the start codon AUG.
3. Elongation: amino acids join one by one by peptide bonds.
4. Termination: at a stop codon, the protein is released.

• UTRs (untranslated regions) are extra parts at both ends of mRNA that are not translated.

Gene regulation: the lac operon

• Proposed by Jacob and Monod in E. coli.
• Regulator gene (i) makes a repressor protein.
• Structural genes:
  • z → β-galactosidase (breaks lactose)
  • y → permease (lets lactose into the cell)
  • a → transacetylase
• Lactose is the inducer. It switches the operon on.
• This is negative regulation, because the repressor switches it off.

Human Genome Project (HGP)

• A huge international project from 1990 to 2003 to read all human DNA.
• Methods: ESTs (Expressed Sequence Tags) to find genes that are expressed, and sequence annotation (read everything, then find what each part does). Vectors used: BAC and YAC. Sequencing method by Frederick Sanger.

DNA fingerprinting

• Developed by Alec Jeffreys.
• Uses VNTRs (Variable Number of Tandem Repeats): short DNA sequences that repeat a different number of times in different people, so everyone's pattern is unique (except identical twins).

1. Isolate DNA (from blood, hair, skin).
2. Cut it with restriction enzymes.
3. Separate the pieces by gel electrophoresis.
4. Transfer them to a nylon membrane (Southern blotting).
5. Hybridise with a labelled VNTR probe.
6. Autoradiography shows the band pattern.

• Uses: solving crimes (forensics), paternity disputes, and studying population diversity and evolution.

30-second revision

• DNA: antiparallel; A=T (2 bonds), G≡C (3 bonds); 10 bp per turn, 3.4 nm pitch.
• Human DNA ≈ 2.2 m long; nucleosome = histone octamer + ~200 bp.
• Griffith → transformation; Avery → DNA; Hershey–Chase (³²P) → DNA is the genetic material.
• Meselson–Stahl → semi-conservative replication.
• Pol I rRNA, Pol II mRNA, Pol III tRNA. Splicing, capping, tailing.
• Stop codons: UAA, UAG, UGA; start: AUG (Met).
• lac operon: z, y, a; lactose = inducer; negative regulation.
• HGP: ~30,000 genes, 99.9% same; DNA fingerprinting uses VNTRs.