Molecular Basis of Inheritance

🧬 Molecular Basis of Inheritance – The Blueprint of Life

1. Discovery of Nucleic Acids & DNA Double Helix

A. Historical Context & Basic Definitions

• Friedrich Miescher (1869): First extracted acidic substance from nucleus of pus cells/owl cells and named it Nuclein.
• Altmann: Proved acidic nature of nuclein and renamed it Nucleic Acid.

  • DNA (Deoxyribonucleic Acid): Genetic material in most organisms; most abundant genetic material.

  • RNA (Ribonucleic Acid): Genetic material in some viruses (TMV, HIV, QB Bacteriophage); acts as messenger, adapter, structural, and catalytic molecule.

B. DNA Length across Organisms

C. Chemical Composition of Nucleotides

• $$\mathbf{\text{Nucleoside} = \text{Pentose Sugar} + \text{Nitrogenous Base}}$$
• $$\mathbf{\text{Nucleotide} = \text{Pentose Sugar} + \text{Nitrogenous Base} + \text{Phosphate Group}}$$
• Nitrogenous Bases:

  • Purines (9-membered double ring; $N$ at 1,3,7,9): Adenine ($A$) and Guanine ($G$).

  • Pyrimidines (6-membered single ring; $N$ at 1,3): Cytosine ($C$), Thymine ($T$ – in DNA), Uracil ($U$ – in RNA).

• Chemical Bonds:

  • N-Glycosidic Linkage: Connects $C_1’$ of sugar with $N_9$ of Purine or $N_1$ of Pyrimidine.

  • Phosphoester Linkage: Connects phosphate group to $C_5’$ OH of nucleoside.

  • $3’\text{–}5’$ Phosphodiester Bond: Connects adjacent nucleotides in a polynucleotide chain.

D. Chargaff’s Rules (Only for dsDNA)

• Molar concentration of Adenine $=$ Thymine ($[A] = [T]$) and Guanine $=$ Cytosine ($[G] = [C]$).
• Total Purines $=$ Total Pyrimidines $\implies \frac{A + G}{T + C} = 1$ (Constant for all species).
• Base Ratio ($\frac{A + T}{G + C}$): Specific for a species. $>1$ in eukaryotes (Human $= 1.52$), $<1$ in prokaryotes ($E. coli = 0.93$).

2. DNA Double Helix Features & Packaging (Nucleosome)

A. Watson & Crick Model of DNA (1953)

Based on X-ray diffraction data of Maurice Wilkins & Rosalind Franklin and Chargaff’s equivalence rules:

• Two polynucleotide chains run in antiparallel polarity ($5′ \to 3’$ and $3′ \to 5’$). Backbone formed by sugar-phosphate, bases project inside.
• Right-handed helical coiling. Diameter $= 20\text{ \AA}\ (2\text{ nm})$.
• Pitch of Helix: $3.4\text{ nm}\ (34\text{ \AA})$ per turn; contains $10\text{ base pairs}$. Distance between adjacent base pairs $= 0.34\text{ nm}\ (3.4\text{ \AA})$.
• Hydrogen Bonding: $A = T$ (2 Hydrogen bonds), $G \equiv C$ (3 Hydrogen bonds). Base stacking provides thermodynamic stability.

B. Formulas for Bond Calculations

C. Packaging of DNA (Nucleosome Model)

• Length of Human DNA: $6.6 \times 10^9\text{ bp} \times 0.34 \times 10^{-9}\text{ m/bp} = \mathbf{2.2\text{ metres}}$.
• Prokaryotes ($E. coli$ DNA length $= 1.36\text{ mm}$): No nucleus, but DNA is held in nucleoid by positively charged non-histone basic proteins.
• Eukaryotic Packaging: Negatively charged acidic DNA wrapped around positively charged basic Histone Octamer (rich in basic amino acids Lysine and Arginine).

  • Histone Octamer Core: Contains 2 copies each of $H_2A, H_2B, H_3, H_4$ (8 proteins).

  • Nucleosome Unit: Octamer core $+$ $H_1$ linker protein $+$ $\mathbf{200\text{ bp}}$ wrapped DNA. Under electron microscope appears as “Beads-on-string” structure.

3. Chromatin Types & Search for Genetic Material

A. Euchromatin vs. Heterochromatin

B. Griffith’s Transformation Experiment (1928)

• Worked on Streptococcus pneumoniae using S-strain (Smooth capsule, Virulent) and R-strain (Rough, Non-virulent):

  • $\text{S-strain} \to \text{Injected into mice} \to \text{Mice Die}$.

  • $\text{R-strain} \to \text{Injected into mice} \to \text{Mice Live}$.

  • $\text{Heat-killed S-strain} \to \text{Injected into mice} \to \text{Mice Live}$.

  • $\text{Heat-killed S-strain} + \text{R-strain} \to \text{Injected into mice} \to \mathbf{\text{Mice Die}}$ (Living S-strain recovered).

• Conclusion: Non-virulent R-strain transformed into virulent S-strain by absorbing a ‘Transforming Principle’.

C. Biochemical Characterisation (Avery, MacLeod, McCarty – 1944)

• Purified proteins, RNA, and DNA from heat-killed S-cells to test transformation:

  • $\text{Proteases} + \text{RNase treatment} \implies \text{Transformation still occurred}$.

  • $\mathbf{\text{DNase treatment}} \implies \mathbf{\text{Transformation inhibited}}$.

• Proved DNA is the transforming genetic material.

D. Hershey & Chase Transduction Experiment (1952)

Unequivocal proof that DNA is genetic material using Bacteriophage $T_2$ and radioactive isotopes:

• Radioactive $\mathbf{^{32}P}$ (Labels DNA): Radioactivity detected in bacterial pellet (cell).
• Radioactive $\mathbf{^{35}S}$ (Labels Protein Coat): Radioactivity detected only in supernatant (viral coats).
• Steps: Infection $\to$ Blending (detached coat) $\to$ Centrifugation. Proved viral DNA enters host cell.

4. DNA vs. RNA & Central Dogma

A. Properties of Genetic Material & DNA vs RNA Comparison

Essential criteria for genetic material: Replication capability, Chemical stability, Scope for slow mutations, and Expression of Mendelian traits.

B. Central Dogma & Reverse Transcription

Proposed by Francis Crick. Describes unidirectional flow of genetic information:

$$\mathbf{\text{DNA}} \xrightarrow{\text{Replication}} \text{DNA} \xrightarrow{\text{Transcription}} \mathbf{\text{RNA}} \xrightarrow{\text{Translation}} \mathbf{\text{Protein}}$$

• Reverse Transcription (Teminism): Discovered by Temin & Baltimore in Rous Sarcoma Virus (RSV). Genetic information flows backwards from $\text{RNA} \to \text{DNA}$ via Reverse Transcriptase enzyme.

5. DNA Replication & Semi-Conservative Proof

A. Semi-Conservative Replication Mechanism

• Proposed by Watson & Crick. Each daughter DNA molecule retains one parental strand and synthesizes one new complementary strand.
• Occurs during S-phase of cell cycle in nucleus (Eukaryotes) or cytoplasm (Prokaryotes).

B. Experimental Proofs

• Meselson & Stahl Experiment (1958):

  • Grew $E. coli$ in heavy nitrogen $\text{N}^{15}$ medium, then transferred to light $\text{N}^{14}$ medium.

  • Centrifuged in $\text{CsCl}$ density gradient.

  • Generation I (20 mins): $100\%$ Hybrid DNA ($\text{N}^{15}\text{-N}^{14}$).

  • Generation II (40 mins): $50\%$ Hybrid ($\text{N}^{15}\text{-N}^{14}$) $+$ $50\%$ Light ($\text{N}^{14}\text{-N}^{14}$).

• Taylor et al. Experiment (1958): Proved semi-conservative replication at chromosomal level in plant Vicia faba (faba beans) using Radioactive Thymidine ($^3\text{H-Thymidine}$).

6. Enzymes of Replication & Y-Shaped Replication Fork

A. Enzymes Involved in Replication

• 1. Helicase: Unwinds double helix by breaking hydrogen bonds (ATP dependent).
• 2. Topoisomerase / DNA Gyrase: Relieves torsional strain and supercoiling ahead of unwinding fork.
• 3. SSBP (Single-Stranded Binding Proteins): Prevents re-annealing of separated single strands.
• 4. Primase (RNA Polymerase): Synthesizes short RNA primer ($6\text{–}10\text{ nucleotides}$) to provide free $3’\text{-OH}$ group.
• 5. DNA Polymerase III: Main polymerizing enzyme; highly efficient ($2000\text{ bp/sec}$ in $E. coli$). Synthesizes DNA strictly in $\mathbf{5′ \to 3′}$ direction.

  • Substrate = Deoxyribonucleoside Triphosphates (dNTPs) (acts as substrate and provides energy via high-energy terminal phosphates).

• 6. DNA Ligase: Joins Okazaki fragments by forming phosphodiester bonds.

B. Leading vs. Lagging Strand Mechanics

7. Gene Types, Transcription Unit & Promoters

A. Gene Types & Split Genes

• Cistron: Segment of DNA coding for a polypeptide chain.

  • Monocistronic: Single gene code per mRNA (Eukaryotes).

  • Polycistronic: Multiple genes/proteins coded from single mRNA (Prokaryotes).

• Split Genes (Eukaryotes): Discontinuous gene containing Exons (coding/expressed sequences) and Introns (non-coding intervening sequences removed during splicing). Non-split genes occur in prokaryotes.

B. Anatomy of Transcription Unit

Comprises 3 regions: Promoter, Structural Gene, Terminator.

• Template Strand ($3′ \to 5’$ Polarity): Strand transcribed into RNA.
• Coding / Sense Strand ($5′ \to 3’$ Polarity): Does not code for RNA, but all reference points (Promoter/Terminator) are defined with respect to its $5’$ and $3’$ ends!

  • Promoter: Located at $5’$-end (upstream) of coding strand; binding site for RNA Polymerase.

  ↳ Prokaryotic Box: Pribnow Box ($TATAAT$).

  ↳ Eukaryotic Box: TATA / Hogness Box (at $-20$ to $-30$) & CAAT Box (at $-70$ to $-80$).

  • Terminator: Located at $3’$-end (downstream) of coding strand; stops transcription.

8. RNA Types & Post-Transcriptional Processing

A. Types of Non-Genomic RNA

B. Eukaryotic RNA Polymerases

• RNA Polymerase I: Synthesizes $28\text{S}, 18\text{S}, 5.8\text{S}$ rRNAs.
• RNA Polymerase II: Synthesizes precursor of mRNA called heterogeneous nuclear RNA (hnRNA).
• RNA Polymerase III: Synthesizes tRNA, $5\text{S}$ rRNA, and snRNA (small nuclear RNA).
• Prokaryotes: Single RNA polymerase ($6\text{ polypeptides}$ core enzyme $+$ $\sigma$ factor) transcribes all RNAs.

C. Post-Transcriptional Modifications (Processing of hnRNA $\to$ mRNA)

1. 1. Capping: Addition of an unusual nucleotide 7-methyl guanosine triphosphate ($7\text{-mG}_{\text{PPP}}$) at $5’$-end of hnRNA. Protects from nucleases and helps ribosome binding.
2. 2. Tailing (Polyadenylation): Addition of 200–300 Adenylate residues (Poly-A tail) at $3’$-end in a template-independent manner.
3. 3. Splicing: Removal of non-coding Introns and joining of coding Exons catalyzed by snRNPs (“Snurps” – small nuclear ribonucleoproteins) forming spliceosome.

9. Genetic Code Features & Codon Mathematics

A. Deciphering the Genetic Code

• George Gamow (Physicist): Proposed that genetic code must be a triplet ($4^3 = 64\text{ codons}$) to code for 20 standard amino acids.
• Har Gobind Khorana: Synthesized RNA molecules with defined combinations of homopolymers and copolymers.
• Marshall Nirenberg: Cell-free system for protein synthesis.
• Severo Ochoa Enzyme (Polynucleotide Phosphorylase): Polymerizes RNA without a template.

B. Salient Features of Genetic Code

• 1. Triplet Nature: 61 codons code for amino acids; 3 codons do not code for any amino acid ($\mathbf{UAA\text{ – Ochre}, UAG\text{ – Amber}, UGA\text{ – Opal}}$ = Stop / Nonsense Codons).
• 2. Initiator Codon: AUG has dual functions: codes for Methionine and acts as start codon.
• 3. Unambiguous & Specific: One particular codon codes for only one specific amino acid.
• 4. Degenerate: Some amino acids are coded by more than one codon (e.g., Leucine, Serine, Arginine).
• 5. Comma-less & Non-overlapping: Read continuously without punctuation.
• 6. Universal: From bacteria to humans, $UUU$ codes for Phenylalanine (Exceptions: Mitochondria & Paramecium).

10. tRNA Adapter Model & Ribosome Subunits

A. Structure of tRNA (Adapter Molecule)

• 2D Secondary Structure (Clover Leaf Model – Robert Holley):

  • $3’$-Acceptor Arm: Contains $CCA\text{-}3’$ sequence; site for amino acid binding (Aminoacyl attachment).

  • Anticodon Loop: Has 7 bases with an anticodon complementary to specific mRNA codon.

  • DHU Loop ($D$-loop): Aminoacyl-tRNA synthetase enzyme binding site.

  • $T\psi C$ Loop: Ribosome attachment site.

  • Variable Loop: Unknown function.

• 3D Tertiary Structure: Compact Inverted L-shaped structure.

B. Ribosome Structure & Translation Sites

Non-membrane bound organelle composed of rRNAs and proteins. Subunits assemble in presence of $\mathbf{Mg^{2+}}$ ions ($0.001\text{ M}$ concentration):

C. Translation Binding Sites on Ribosome

• A-site (Aminoacyl site): Accepts incoming charged tRNA.
• P-site (Peptidyl site): Holds tRNA carrying growing polypeptide chain; site where peptide bond is formed.
• E-site (Exit site): Site from where uncharged empty tRNA leaves ribosome.

11. Translation Mechanism & Energy Requirements

A. Overview & Essential Energy Components

Translation is the process of polymerizing amino acids into a polypeptide chain directed by mRNA sequence. Occurs in 4 distinct steps:

B. Step 1: Amino Acid Activation & tRNA Charging

Reaction cascade occurring in cytoplasm before ribosome assembly:

$$\text{Amino Acid} + \text{ATP} \xrightarrow[\text{Mg}^{2+}]{\text{Aminoacyl-tRNA Synthetase}} \text{Aminoacyl-AMP-Enzyme Complex} + PP_i$$
$$\text{Complex} + \text{tRNA} \to \mathbf{\text{Aminoacyl-tRNA (Charged tRNA)}} + \text{AMP} + \text{Enzyme}$$

12. Initiation, Elongation & Termination Steps

A. Step 2: Initiation & Shine-Dalgarno Sequence

• $IF_1$ & $IF_3$ bind to $30\text{S}$ small ribosomal subunit.
• Small subunit binds to mRNA at Untranslated Region (UTR) via the Shine-Dalgarno Sequence ($5’\text{-AGGAGG-}3’$ in prokaryotic mRNA complementary to $16\text{S rRNA}$ Anti-Shine Dalgarno sequence $3’\text{-UCCUCC-}5’$).
• Initiating Amino Acid:

  • Prokaryotes: N-Formyl Methionine (f-Met).

  • Eukaryotes & Archaea: Methionine (Met).

• Initiator tRNA carrying f-Met with anticodon $3’\text{-UAC-}5’$ binds to start codon $5’\text{-AUG-}3’$ directly at P-site (Peptidyl site).
• Large $50\text{S}$ subunit joins forming $70\text{S}$ Initiation Complex ($IF$ factors released).

B. Step 3: Elongation & Translocation

• Next charged tRNA enters empty A-site (Aminoacyl site) driven by Elongation Factors $+$ GTP.
• Peptide bond formed between amino acids at P & A sites by enzyme Peptidyl Transferase ($23\text{S rRNA}$ ribozyme in prokaryotes / $28\text{S rRNA}$ in eukaryotes; non-proteinaceous catalytic RNA).
• Translocation: Ribosome moves 1 codon ahead along mRNA in $5′ \to 3’$ direction. Uncharged tRNA shifts to E-site and leaves; growing peptidyl-tRNA shifts from A-site to P-site.

C. Step 4: Termination

• When a Stop / Nonsense Codon ($UAA, UAG, UGA$) reaches A-site, no tRNA can recognize it.
• Release Factors (RF) bind to stop codon:

  • $RF_1$: Recognizes $UAA$ & $UAG$.

  • $RF_2$: Recognizes $UAA$ & $UGA$.

  • $RF_3$: Helps $RF_1 / RF_2$ release.

• Polypeptide chain is cleaved, tRNA is released, and entire $70\text{S}$ ribosomal machinery dissociates.

13. Regulation of Gene Expression

A. Levels of Gene Regulation in Eukaryotes

Can be regulated at 4 distinct organizational levels:

1. Transcriptional Level: Formation of primary transcript / hnRNA (Primary control point).
2. Processing Level: Post-transcriptional modifications (Splicing, Capping, Tailing).
3. Transport Level: Transport of mature mRNA from nucleus into cytoplasm.
4. Translational Level: Polypeptide protein synthesis at ribosome.

B. Levels of Gene Regulation in Prokaryotes

• Predominantly controlled at the Transcriptional Initiation level (e.g., Operon models like Lac Operon).
• Translational control also occurs, but since transcription and translation are coupled in prokaryotic cytoplasm, transcriptional regulation is primary.