Molecular Biology Comprehensive Notes

LCourse & Examination Logistics

  • Molecular Biology module: 3 CFU (within 14 CFU Biochemistry course)

    • Exam taken only after passing Biochemistry (written + oral)

    • Registration via MS-Teams (not Delphi)

    • Skipping an exam attempt cancels previous grade

  • Immediate post-course written MCQ test (May)

  • Allowed texts: Lehninger, Watson; class notes highly recommended

  • Validation of prior Molecular Biology credits:

    • Email syllabus + certificate to Prof. Piro ➔ approval email to class reps

Protein Generalities

  • Functions: transport (e.g. hemoglobin), enzymes (lower EaE_a, metabolic control), reserve, regulation, structural (e.g.

  • 3-D shape ⇒ function; DNA is structurally uniform, proteins are not

  • Collagen: most abundant animal protein (extracellular, fibrous)

  • Keratin: intracellular (cytoskeleton), fibrous

  • Fibrous vs. globular:

    • Fibrous: repetitive sequence, insoluble, structural support

    • Globular: irregular sequence, soluble, transport / enzymatic, pH-sensitive

Amino Acids (20 standard)

Classification (R-groups)

  • Non-polar (hydrophobic): aliphatic side chains; Met encoded by AUGAUG, always 1st residue; Pro cyclic, breaks α-helix; Gly small

  • Aromatic: Phe, Tyr (–OH ⇒ phosphorylation), Trp; absorb UV (protein quantitation)

  • Uncharged polar: Ser, Thr (both phosphorylatable), Cys (forms disulfide bridge), Asn, Gln

  • Positively charged: Lys (ϵ\epsilon-NH₃⁺), Arg (guanidinium), His (charged < pH 6; epigenetic hot-spot in histones)

  • Negatively charged: Asp, Glu (extra –COO⁻)

Peptide Bond

  • Amide between –COOH & –NH₂; partial double-bond character ⇒ planar, no rotation; rotation only around ϕ,ψ\phi,\psi of CαC_\alpha

  • Condensation eliminates H2OH_2O; proteins have free N-ter & C-ter (e.g. histone N-ter tails)

Structural Hierarchy

  • Primary: AA sequence from mRNA codons

  • Secondary: recurrent H-bonded motifs, sequence-independent dimensions

    • α-helix: 3.6  aa/turn3.6\;aa/turn, right-handed, H-bond i→i+4i\rightarrow i+4; broken by Pro/Gly

    • β-sheet: antiparallel/parallel, side chains alternate ↑↓

    • Loops/coils connect elements; flexibility for substrate fit

  • Tertiary: long-range folding via H-bonds, ionic, hydrophobic, Van der Waals, disulfide bridges; denatured by heat, urea, extreme pH

  • Quaternary: spatial arrangement of ≥2 subunits (e.g. α<em>2β</em>2\alpha<em>2\beta</em>2 hemoglobin)

  • Native conformation: only functional form; chaperones (Hsp) aid folding; mis-fold ⇒ loss of function

DNA & RNA Foundations

Historical Experiments

  • Griffith (1928): heat-killed S strain transforms live R ➔ virulence factor

  • Avery et al. (1944): DNA identified as “transforming principle”

B-DNA Parameters

  • Helix pitch: 3.4 A˚3.4\,\text{Å}, 10.510.5 bp/turn; width 20 A˚20\,\text{Å}

  • Major groove ≈ 22 Å, minor ≈ 12 Å ⇒ TFs bind major groove (α-helix fits)

  • Antiparallel strands, 5′-P / 3′-OH polarity

  • Base pairing: A!:!TA!:!T (2 H-bonds), G!:!CG!:!C (3); hydrophobic base stacking stabilises helix

  • Alternative forms: A- (11 bp/turn), Z-DNA; conformational change alters topology

Unusual DNA Structures

  • Palindromes, mirror repeats; triplex (Hoogsteen), hairpins/cruciforms, G-quadruplex (G-rich)

  • Denaturation/Renaturation: TmT_m ↑ with GC%, length, [salt]; basis for PCR, hybridisation

DNA Topology

  • Linking number LK=bp10.5LK=\frac{\text{bp}}{10.5} (relaxed B-DNA)

  • ΔLK ≠ 0 ⇒ supercoiling; negative (under-wound) common (σ ≈ –0.06 in bacteria)

  • Forms: plectonemic (free DNA) vs. solenoidal (nucleosome wrapping)

  • Topoisomerases:

    • Type I: cut 1 strand, ΔLK ±1, ATP-independent (relaxation)

    • Type II (DNA gyrase, euk. topo II): cut both strands, ΔLK ±2, ATP-dependent; target of antibiotics (nalidixic acid, ciprofloxacin) & chemotherapeutics (doxorubicin, etoposide)

RNA Variety & Modifications

  • mRNA (cap-5′, poly-A-3′, introns removed), rRNA (97 % of cellular RNA), tRNA, snRNA, snoRNA, miRNA, siRNA, piRNA, lncRNA, circRNA

  • tRNA: 75-95 nt, cloverleaf → L-shape; CCA-3′ added post-transcriptionally; modified bases > 100 (Ψ, D, Inosine at wobble position)

    • Aminoacyl-tRNA synthetases (20): charge tRNA; proofreading via editing site; energy from ATP→AMPATP\rightarrow AMP

  • rRNA: catalytic ribozyme; 16S (decoding), 23S/28S (peptidyl-transferase), 5S

Translation Mechanics

Bacterial Initiation

  • 30S + IF-1 (blocks A), IF-3 (prevents 50S joining), IF-2-GTP + fMet-tRNA bind Shine-Dalgarno (RBS) → position AUG in P-site

  • 50S joins, GTP hydrolysed ⇒ 70S initiation complex

Elongation Cycle

  1. EF-Tu-GTP delivers aa-tRNA to A-site; correct codon–anticodon ⇒ GTP→GDP & release

  2. Peptidyl transferase (23S) moves polypeptide to A-site tRNA

  3. EF-G-GTP translocates ribosome (A→P→E); EF-Ts regenerates EF-Tu-GTP

Termination & Recycling

  • Stop codon (UAA, UAG, UGA) recognised by RF-1/2; RF-3-GTP releases RF

  • RRF + EF-G disassemble ribosome; IF-3 binds 30S to prevent premature reassociation

Eukaryotic Differences

  • 40S binds eIF-4E (cap), eIF-4G, PABP (poly-A) ⇒ mRNA circularisation; scans to first Kozak-AUG; initiator Met-tRNA (no formyl)

  • More initiation factors; elongation/termination essentially conserved

  • mTOR kinase phosphorylates 4E-BP → frees eIF-4E ⇒ translation ON; inhibited by rapamycin

Protein Folding & Quality Control

  • Chaperones (Hsp70, Hsp60, Hsp90) assist co- & post-translational folding

  • Misfold/aggregation triggers ubiquitin-proteasome degradation or ER stress response

  • Proteasome: ATP-dependent, highly regulated; extracellular proteases uncontrolled (digestion)

Chromatin Architecture & Epigenetics

Nucleosome

  • Core: 2×(H2A H2B H3 H4) + 147 bp DNA (1.65 turns)

  • Linker DNA (20-80 bp) + H1 → 30 nm fibre (solenoid or zig-zag)

  • Histone tails (N-ter) subject to covalent PTMs: acetyl (HAT/HDAC), methyl (HMT/HDM), phospho, ubiquitin

  • Histone variants: H2A.Z, H2A.X (Ser-139 P marks DSB), H3.3, CENP-A

  • Chromatin states: euchromatin (open), heterochromatin (constitutive vs. facultative); regulated by PTMs & DNA methylation (CpG)

  • Higher order: loops (~90 kb) anchored by cohesin, CTCF forming TADs; compartments A (active) / B (inactive); chromosome territories

DNA Methylation & Disease

  • DNMT-1 (maintenance, somatic), DNMT-3A/B/L (de novo, germline/embryo)

  • CpG hypermethylation of promoters ⇒ gene silencing (tumour suppressors)

  • Global hypomethylation + focal hypermethylation in cancer; Rett syndrome: MECP2 mutations disrupt methyl-CpG binding

Telomeres & Senescence

  • Telomere repeat TTAGGGnTTAGGG_n, 5–20 kb; 3′ overhang protected by shelterin (TRF1/2, POT1…)

  • End-replication problem solved by telomerase (TERT + TERC RNA template)

  • Telomere shortening ⇒ DNA damage response (ATM/ATR → p53/p21 or p16) ⇒ senescence or apoptosis

  • Telomere syndromes: Dyskeratosis congenita (TERC, DKC1), Werner, Bloom, pulmonary fibrosis; anticipation: telomere length decreases across generations

Cell Cycle Control

  • Phases: G₁ → S → G₂ → M; quiescent G₀; checkpoints: G₁ (restriction), G₂/M, spindle (M)

  • Cyclin-CDK complexes drive transitions; inhibited by CKIs (p21^Cip1, p27^Kip1, p16^Ink4a)

  • Rb binds E2F; phosphorylation by Cyclin D-CDK4/6 releases E2F → S-phase genes

  • DNA damage: ATM/ATR phosphorylate p53 → p21 ↑ → Cyclin-CDK inhibited → arrest/repair; p53 loss common in tumours

DNA Damage & Repair

  • Spontaneous lesions: depurination (10⁴/cell/day), deamination (C→U, A→I), ROS oxidation (8-oxo-dG)

  • Photodimers (UV): repaired by photolyase (prokaryotes) or NER (humans)

  • Base Excision Repair (BER): glycosylase removes base → AP endonuclease → Pol β → ligase

  • Nucleotide Excision Repair (NER): UvrABCD (prok) / XPA-G, TFIIH (euk); defects ⇒ Xeroderma pigmentosum, Cockayne, TTD

  • Mismatch Repair (MMR): MutS/L/H (prok); MSH/MLH (euk); reduces error 10²-fold.

  • Double-strand break repair:

    • Homologous Recombination (HR): RAD51, BRCA1/2; error-free

    • Non-Homologous End-Joining (NHEJ): KU70/80, DNA-PKcs, Artemis, Ligase IV; error-prone

    • TLS polymerases (Pol IV/V, ζ, η, κ) bypass lesions; introduces mutations; SOS response

Molecular Biology Techniques

  • Restriction enzymes (palindromic sites) + ligase → recombinant plasmids; essential features: ori, polylinker (MCS), selectable marker (Amp^R, Tet^R)

  • Transformation (bacteria) / transfection (eukaryotes); antibiotic selection maintains plasmid

  • Fusion tags (GST, His₆) enable affinity chromatography purification (ligand: glutathione, Ni²⁺)

  • Site-directed mutagenesis: oligo with desired base change; PCR or heteroduplex selection; Dpn I digests methylated parental DNA

  • PCR: denature 95 °C, anneal 50-65 °C, extend 72 °C; 2n2^n amplification; Taq polymerase (thermostable)

    • qPCR (Real-Time): SYBR Green fluorescence; Ct value inversely proportional to log [template]

  • Reverse-Transcriptase PCR: mRNA → cDNA (intron-free) for cloning/expression

  • Sanger sequencing: ddNTP chain-termination; capillary electrophoresis with dye-labelled terminators; basis for Human Genome (2001)

  • Next-Gen Sequencing (NGS): massively parallel short reads; hours vs. years

RNA Interference & miRNA

  • Dicer processes dsRNA → 21-22 nt siRNA; loaded into RISC (Ago) → mRNA cleavage/silencing

  • Endogenous pri-miRNA → Drosha → pre-miRNA → Dicer; guide strand targets 3′-UTR (imperfect pairing ⇒ translation block; perfect ⇒ mRNA decay)

  • miRNA regulate ~60 % of genes (≈ 2000 human miRNA); dysregulation in cancer (miR-21 oncogenic; miR-34 tumour suppressor)

  • Experimental gene knock-down: synthetic siRNA or shRNA vectors; transient, sequence-specific

Long Non-Coding & Circular RNAs

  • lncRNA > 200 nt; roles: chromatin scaffold (XIST silences X-chromosome), decoy, guide, sponge (ceRNA)

  • circRNA: covalently closed; stable; act as miRNA sponges, RBP binders

. Genomic Imprinting & Disorders

  • ~100 genes monoallelically expressed depending on parent; controlled by Imprint Control Regions (ICR) via DNA methylation

  • Examples on chr 11p15 (IGF2 paternal, H19 maternal):

    • Beckwith-Wiedemann (BWS): maternal ICR hypermethylation → IGF2 biallelic → overgrowth, tumor risk

    • Russell-Silver (RSS): paternal ICR hypomethylation → IGF2 under-expression → growth retardation

  • Other imprinting disorders: Prader–Willi, Angelman (chr 15), involve deletions, UPD, methylation defects

(End of comprehensive bullet-point notes)