AI notes Topic 2

1. DNA AS GENETIC MATERIAL

Griffith's transformation experiment (1928)

Showed heat-killed S-strain bacteria could transform R-strain into virulent form (DNA was transferable factor)

Avery-MacLeod-McCarty experiment (1944)

Proved DNA (not protein) was the transforming principle using enzyme treatments

Hershey-Chase experiment (1952)

Confirmed DNA as genetic material using radioactive phage (32P-DNA entered bacteria, not 35S-protein)

2. DNA STRUCTURE

Chargaff's rules

A=T, C=G; purines = pyrimidines

Watson-Crick model (1953)

Double helix with antiparallel strands, H-bonds between bases (A-T, C-G)

Rosalind Franklin's contribution

X-ray diffraction showed helical structure with 2nm diameter

3. DNA REPLICATION

Semiconservative replication

Each new DNA molecule has 1 parental + 1 new strand (Meselson-Stahl experiment)

Leading vs. lagging strand

Leading: continuous 5’→3’; Lagging: Okazaki fragments (discontinuous)

Key bacterial enzymes

Helicase (unwinds), SSB proteins (stabilize), Primase (RNA primer), DNA Pol III (elongates), DNA Pol I (replaces primer), Ligase (joins fragments)

4. EUKARYOTIC REPLICATION

Telomeres

TTAGGG repeats at chromosome ends; shortened each replication (except in germ cells/telomerase-active cells)

Telomerase function

Adds telomeric repeats using RNA template; prevents chromosome shortening

Hayflick limit

~50-70 cell divisions before telomere shortening triggers apoptosis

5. TRANSCRIPTION (BACTERIA)

Promoter elements

-35 box (TTGACA) and -10 Pribnow box (TATAAT)

RNA polymerase subunits

α, β, β’, ω, σ (sigma factor recognizes promoter)

Termination types

Rho-dependent (requires Rho protein) vs. Rho-independent (GC-rich stem-loop + poly-U)

6. TRANSCRIPTION (EUKARYOTES)

RNA Polymerases

Pol I (rRNA), Pol II (mRNA, snRNA), Pol III (tRNA, 5S rRNA)

Pre-initiation complex (PIC)

TFIID binds TATA box → recruits Pol II + other TFs (TFIIA, B, E, F, H)

mRNA processing

5’ cap (7-methylguanosine), poly-A tail, splicing (snRNPs remove introns)

7. GENETIC CODE

Codon properties

Triplet (3 nucleotides), non-overlapping, degenerate (64 codons → 20 AAs)

Start codon

AUG (Met); Stop codons

Wobble hypothesis

3rd base in codon can pair flexibly (e.g., tRNA anticodon 5’-UCC-3’ reads AGU/AGC)

8. TRANSLATION

Ribosome sites

A (aminoacyl-tRNA), P (peptidyl-tRNA), E (exit)

Initiation (Bacteria)

Shine-Dalgarno sequence aligns 30S subunit; fMet-tRNA binds P site

Elongation

EF-Tu delivers aminoacyl-tRNA to A site; peptidyl transferase forms peptide bond

Termination

Release factors (RF1/2 in bacteria, eRF1 in eukaryotes) recognize stop codons

9. MUTATIONS

Point mutation types

Substitution (transition: purine→purine; transversion: purine↔pyrimidine), insertion, deletion

Frameshift mutation

+1/-1 bp indels alter reading frame → premature stop or nonfunctional protein

Missense vs. nonsense

Missense: AA change; Nonsense: stop codon introduced

Silent mutation

Codon changes but same AA (synonymous)

10. MUTATION EXAMPLES

Ras G12V mutation

Gly→Val at position 12 locks Ras in active state → cancer

β-hemoglobin mutations

Promoter (reduced transcription), splicing (intron retention), cryptic splice sites (AG creation)

Cystic fibrosis ΔF508

Deletion of Phe508 in CFTR → misfolded protein

11. NONCODING MUTATIONS

Promoter mutations

Alter transcription efficiency (e.g., β-globin promoter → thalassemia)

Splice site mutations

GU-AG rule violation → exon skipping or intron retention (e.g., β-globin IVS-110 G→A)

Poly-A signal mutations

AAUAAA alteration → defective mRNA processing/degradation