Molecular Basis of Inheritance

The DNA

Chapter Focus

• This chapter explains the molecular basis of inheritance including DNA structure, genetic material, replication, transcription, translation, gene regulation, lac operon, Human Genome Project, and DNA fingerprinting.

DNA: The Blueprint of Life

Introduction to DNA

• Mendel explained inheritance patterns, but the physical material responsible for inheritance was unknown at that time.
• Later, DNA (Deoxyribonucleic Acid) was identified as the genetic material in most organisms.
• DNA and RNA are nucleic acids made of nucleotides.

DNA and RNA

• DNA:
  • Acts as the genetic material in most organisms.
• RNA:
  • Acts as genetic material in some viruses.
  • Functions as messenger (mRNA), adapter (tRNA), structural (rRNA), and sometimes catalytic molecule.

DNA as a Polymer

• Definition
  • DNA is a long chain (polymer) of nucleotides (deoxyribonucleotides).
• Length of DNA
  • Defined by number of nucleotides or base pairs (bp).
  • Length is species-specific.
  • Examples:
    • Human haploid genome: 3.3 × 10⁹ bp
    • Escherichia coli: 4.6 × 10⁶ bp
    • Bacteriophage lambda: 48,502 bp
    • Bacteriophage φX174: 5,386 nucleotides

Structure of Polynucleotide Chain

Components of a Nucleotide

1. Nitrogenous Base: Two types:

1. Purines
   • Adenine (A)
   • Guanine (G)
2. Pyrimidines
   • Cytosine (C)
   • Thymine (T) – present in DNA
   • Uracil (U) – present in RNA instead of Thymine

• Base Pairing
  • A pairs with T (2 hydrogen bonds)
  • G pairs with C (3 hydrogen bonds)
• Chargaff’s Rule
  • A = T
  • G = C
  • A + G = T + C
  • A + T / G + C ratio varies between species but is constant within a species.

2. Pentose Sugar

1. Deoxyribose in DNA
2. Ribose in RNA
   • RNA has an additional –OH group at 2′ carbon.

3. Phosphate Group

• Nucleoside
  • Base + Sugar (N-glycosidic bond at 1′ carbon)
• Nucleotide
  • Nucleoside + Phosphate (attached at 5′ carbon)

• Phosphodiester Bond
  • Two nucleotides are linked by 3′–5′ phosphodiester linkage.
  • Forms sugar-phosphate backbone.

Polarity of DNA Strand

• Each strand has directionality:
  • 5′ end → phosphate group free
  • 3′ end → hydroxyl group free

Discovery of DNA

• 1869 – Friedrich Meischer
  • Discovered DNA and named it “Nuclein”.

• 1953 – Watson and Crick
  • Proposed Double Helix Model.
• Based on:
  • X-ray diffraction data (Rosalind Franklin)
  • Chargaff’s observations
  • Maurice Wilkins’ work

Double Helix Structure of DNA

• Strands
  • Two polynucleotide strands.
• Backbone
  • Sugar-phosphate backbone on outside.
• Bases
  • Nitrogen bases project inside.
• Antiparallel Nature
  • One strand runs 5′ → 3′
  • Other runs 3′ → 5′
• Complementary Base Pairing
  • Purine always pairs with pyrimidine.
  • Maintains uniform width of helix.
• Helical Structure
  • Right-handed double helix.
• Dimensions
  • 10 base pairs per turn
  • Helix pitch: 3.4 nm
  • Distance between two base pairs: 0.34 nm
• Stability
  • Hydrogen bonds between bases
  • Base stacking interactions add stability

Packaging of DNA Helix

DNA Length and Packaging Problem

• Why Packaging is Needed
  • In a typical human cell, DNA length ≈ 2.2 meters
  • Nuclear diameter ≈ 10⁻⁶ meters
• Question
  • How is 2.2 m long DNA packed inside such a tiny nucleus?

Thus, extremely efficient packaging is required without disturbing gene function.

DNA Packaging in Prokaryotes

Example: Escherichia coli

• DNA Length
  • ≈ 1.36 mm.
• Packaging Method
  • DNA is supercoiled (coiled and recoiled to form large loops).
  • DNA (negative charge) is held by proteins (positive charge).
• Proteins Involved
  • Non-histone basic proteins
  • Polyamines
  • RNAs
• Region
  • Compacted DNA mass is called nucleoid.
  • or DNA is held in a region called the nucleoid.
• Key Point
  • No histones present in prokaryotes (as per NCERT level).

DNA Packaging in Eukaryotes

• Complexity
  • Eukaryotic DNA packaging is more organized and complex than in prokaryotes.
  • DNA is wrapped around proteins called histones.
• Main Proteins Involved
  • Histones
• Histone Characteristics
  • Rich in basic amino acids: Lysine and Arginine
  • Positively charged due to these amino acids.
• Packaging
  • Histones are positively charged.
  • DNA is negatively charged (due to phosphate groups)
  • Electrostatic attraction between positive histones and negative DNA enables tight packaging.

Histones and Nucleosome

• Types of Histones (not mentioned in NCERT)
  • H2A
  • H2B
  • H3
  • H4
  • H1
• Core Histones: H2A, H2B, H3, H4
• Histone Octamer: Two molecules each of:
  • H2A, H2B, H3, H4
  • Total = 8 histone proteins
• Histones form a unit of eight molecules called a histone octamer.

Note- H1 is not part of the octamer.

Nucleosome Formation

• DNA wraps around the histone octamer, forming a nucleosome.
• Wrapping Pattern
  • Approximately 1.65 turns of DNA around the octamer.
  • DNA Length per Nucleosome: ≈ 200 base pairs (including linker region).
  • Core DNA around octamer ≈ 146 bp.
• Definition: A nucleosome = Histone octamer + wrapped DNA
• Appearance: Under electron microscope → “Beads-on-a-string”.
  • Beads = Nucleosomes
  • String = Linker DNA

Chromatosome (not mentioned in NCERT)

• Linker DNA
  • DNA segment connecting two nucleosomes.
• H1 Histone
  • Binds to linker DNA
  • Stabilizes nucleosome structure
  • Acts as a locking/plugging protein
• Definition: Nucleosome + Linker DNA + H1 = Chromatosome

Chromatin

• Definition
  • Nucleosomes collectively form chromatin.
• Structure/Appearance
  • Thread-like stained bodies visible in nucleus during interphase.

Types of Chromatin

1. Euchromatin
   • Loosely packed
   • Lightly stained
   • Transcriptionally active
   • Genes are accessible
   • Replication in early S-phase
2. Heterochromatin
   • Densely packed
   • Darkly stained
   • Transcriptionally inactive
   • Gene expression minimal
   • Replication in late S-phase

Further Higher Level Packaging

• Chromatin fibers are further coiled and condensed to form chromosomes during metaphase.
  • Step 1: Beads-on-string fold into 30 nm fiber.
  • Step 2: Further loop formation and supercoiling.
  • Step 3: Maximum condensation during metaphase.
• Result: Fully condensed chromosomes visible during cell division.
• Additional Proteins
  • Non-histone chromosomal (NHC) proteins
    • Help in higher-order folding
    • Involved in gene regulation
    • Provide structural support

The Search for Genetic Material

• The journey to discover DNA as the genetic material took a long time.
• Initially, it was unclear whether genetic material was:
  • DNA or Protein.

Transforming Principle – Griffith Experiment (1928)

• Scientist
  • Frederick Griffith.
• Organism used
  • Streptococcus pneumoniae (Bacteria causing pneumonia).
• Two Types of Strains
  1. S Strain (Smooth)
     • Smooth colony appearance
     • Surrounded by polysaccharide capsule
     • Virulent (causes disease)
     • Protects bacteria from host immune system
  2. R Strain (Rough)
     • Rough colony appearance
     • No capsule
     • Non-virulent
     • Easily destroyed by host immune cells

Experimental Observations

1. Live S strain injected into mice
   • → Mice died
   • → S bacteria recovered from dead mice
2. Live R strain injected into mice
   • → Mice lived
3. Heat-killed S strain injected into mice
   • → Mice lived
   • → Dead bacteria alone cannot cause disease
4. Heat-killed S strain + Live R strain injected
   • → Mice died
   • → Live S bacteria recovered from dead mice

Critical Observation

• Live R bacteria were transformed into virulent S type bacteria.
• This means: Some substance from dead S cells converted R cells into S cells.

Conclusion

• There exists a “Transforming Principle”
  • It transferred virulence
  • It changed genetic characteristics of R strain
  • It was inheritable
• However, Nature of the transforming principle was unknown at that time.

Avery, MacLeod and McCarty Experiment (1933–44)

• Objective
  • To identify the biochemical nature of Griffith’s “transforming principle”.
• Background
  • Griffith showed that some substance from heat-killed S strain converted R strain into virulent S strain.
  • But the chemical nature of this substance was unknown.

Experimental Approach

• Scientists
  1. Oswald Avery
  2. Colin MacLeod
  3. Maclyn McCarty
• Material Used
  • Heat-killed virulent S strain of Streptococcus pneumoniae.

Procedure

• Step 1: Extracted and purified different biochemical components from heat-killed S cells:
  • Proteins
  • RNA
  • DNA
• Step 2: Each component was separately mixed with live R strain bacteria.
• Step 3: Specific enzymes were used to destroy individual components:
  • Protease → destroys proteins
  • RNase → destroys RNA
  • DNase → destroys DNA

Results

• Protease Treatment → Proteins destroyed
  • → Transformation still occurred
  • → R strain converted into virulent S strain
• RNase Treatment → RNA destroyed
  • → Transformation still occurred
• DNase Treatment → DNA destroyed
  • → Transformation stopped
  • → R strain did NOT convert into S strain

• Conclusion
  • DNA is the transforming principle.
  • DNA carries hereditary information.
  • Proteins and RNA are not the genetic material in this case.
• Significance
  • This was the first direct experimental evidence that DNA is the hereditary material.

Hershey–Chase Experiment (1952)

• Objective
  • To confirm whether DNA or protein is the genetic material.
• Scientists
  • Alfred Hershey and Martha Chase.
• Organism taken for experiment
  • Bacteriophage (virus) infecting Escherichia coli.
• Reason for Using Bacteriophage: Phage consists only of:
  • DNA core
  • Protein coat
  • Thus ideal to test whether DNA or protein enters bacteria.

Experimental Method: Radioactive labeling

1. DNA Labeling
   • With radioactive phosphorus (³²P)
   • Reason: DNA contains phosphorus but no sulfur.
2. Protein Labeling
   • Radioactive sulfur (³⁵S)
   • Reason: Proteins contain sulfur but DNA does not.

• Two Separate Phage Cultures Prepared
  • One with radioactive DNA (³²P)
  • One with radioactive protein (³⁵S)

• Procedure
  • Allowed labeled phages to infect E. coli.
  • Used blender to separate phage coats from bacteria.
  • Centrifuged mixture to separate:
    • Pellet → Bacteria
    • Supernatant → Phage coats

Observation

• ³²P-labeled DNA
  • Radioactivity detected inside bacterial cells (pellet).
• ³⁵S-labeled protein
  • Radioactivity remained outside (supernatant).

• Conclusion
  • Only DNA entered the bacterial cells.
  • Protein coat did not enter the bacterial cells.
  • Therefore, DNA is the genetic material.
• Significance
  • Provided conclusive proof that DNA carries genetic information.

Properties of Genetic Material

• Criteria for Genetic Material
  1. Replication: Must produce its own copies.
  2. Stability: Must be chemically and structurally stable.
  3. Mutation: Must allow slow changes (mutations) for evolution.
  4. Expression: Must express itself in form of traits or Mendelian Characters.

Replication: Both DNA and RNA can replicate due to base pairing.

DNA vs RNA as Genetic Material

Feature	DNA	RNA
Stability	More stable chemically and structurally	Less stable.
	Thymine presence & 2′-OH group absence adds stability	2′-OH group makes RNA unstable/reactive
Chemical Reactivity	DNA less reactive.	RNA more reactive and catalytic.
Base Difference	DNA contains thymine.	RNA contains uracil.
Mutation Rate	DNA mutates slower.	RNA mutates faster → rapid evolution in RNA viruses.
Expression	DNA depends on RNA for protein synthesis.	RNA can directly code for proteins.
Conclusion	DNA is better suited for long-term genetic storage.	RNA is better suited for transmission and rapid evolution.

RNA World

Which Came First: DNA or RNA?

• RNA was the first genetic material.
• Scientific Evidence: RNA played key roles in essential life processes like metabolism and protein synthesis.

RNA in Early Life

• RNA performed dual roles:
  1. Genetic Material:
     • RNA stored genetic information.
     • Even today, some viruses use RNA as genetic material.
  2. Catalyst:
     • RNA acted as an enzyme (ribozymes).
     • Because RNA was catalytic, it was chemically reactive and unstable.

Why DNA Evolved from RNA

• Problems with RNA
  • Presence of 2′-OH group makes RNA chemically reactive.
  • Reactive nature makes RNA unstable.
• Evolution of DNA
  • DNA evolved from RNA with chemical modifications that made it more stable.

• Reasons DNA is More Stable
  • Double-stranded structure
  • Complementary base pairing
  • Absence of 2′-OH group
  • Presence of thymine instead of uracil
  • DNA repair mechanisms

• Conclusion
  • DNA became the preferred genetic material due to structural and chemical stability.

Key Points:

• RNA was crucial in early life for both genetic information and catalysis.
• DNA evolved from RNA to provide a more stable and reliable form of genetic material.

Note- Today RNA functions mainly in expression of genetic information.

Types of Cellular RNA (Non-genetic RNA)

Based on molecular size and function:

1. mRNA (Messenger RNA)

• Percentage:
  • About 3.5% of cellular RNA
• Function:
  • Carries genetic information from DNA to ribosome.
  • Acts as template for protein synthesis.
• Feature:
  • Usually short-lived and unstable.

2. tRNA (Transfer RNA)

• Percentage:
  • ≈ 10–15% of total cellular RNA.
• Other Names:
  • Soluble RNA (sRNA)
  • Adapter RNA
• Function:
  • Brings specific amino acids to ribosome.
  • Decodes genetic information.
• Structure:
  • Clover-leaf secondary structure
  • Anticodon loop → binds to codon on mRNA
  • Amino acid acceptor end → binds specific amino acid
• Special Feature:
  • Each tRNA is specific to one amino acid.
  • No tRNA exists for stop codons.

3. rRNA (Ribosomal RNA)

• Percentage:
  • ≈ 80% of total cellular RNA.
• Function:
  • Structural component of ribosome
  • Catalyzes peptide bond formation
  • Acts as ribozyme (enzyme-like RNA)
• Site of Formation:
  • Synthesized in nucleolus (in eukaryotes).

Central Dogma

• Proposed by Francis Crick
  • Describes the flow of genetic information within a biological system.
  • Statement
    • Genetic information flows in one direction: DNA → RNA → Protein.

Steps in Central Dogma

1. Transcription
   • The process where DNA is used as a template to make RNA, i.e., DNA → RNA.
   • Occurs in nucleus (in eukaryotes).
2. Translation
   • The process where RNA is used to build proteins, i.e., RNA → Protein
   • Occurs on ribosomes in cytoplasm.

Note- The Central Dogma also encompasses DNA Replication (DNA → DNA), which must occur before the transcription and translation stages.

Important Molecules in Central Dogma

• DNA
  • Stores genetic blueprint of an organism.
• mRNA (Messenger RNA):
  • Carries genetic information from DNA to the ribosome.
• tRNA (Transfer RNA):
  • Brings amino acids to ribosome to build proteins.
• rRNA (Ribosomal RNA):
  • Forms the core of the ribosome’s structure and catalyzes protein synthesis.

Exception to Central Dogma

• Reverse Transcription
  • RNA → DNA
• Occurs in some viruses.
• Called Reverse Transcription or Reverse Central Dogma.

Energy Source

• Uses Deoxyribonucleoside Triphosphates (dNTPs), which also provide energy for the reaction.
  • Energy released from breakdown of triphosphates (like ATP) drives polymerization.

Replication Process

• Definition:
  • DNA unwinds at a small section (opening) called the replication fork.
  • Replication fork is the Y-shaped region where DNA is actively replicating.
• Key Concept:
  • DNA does NOT open completely at once due to high energy requirement.
  • Instead, opening and replication progresses gradually in both directions.
• Directionality: DNA polymerase works in one direction (5′ to 3′), creating continuous synthesis on one strand and discontinuous synthesis on the other.
  • Okazaki Fragments: Short segments made on the discontinuous strand are later joined by DNA ligase.

Important Enzymes in Replication

• Helicase
  • Unwinds double helix at origin.
  • Breaks hydrogen bonds between base pairs.
• Single-Stranded Binding Proteins (SSBPs)
  • Bind to separated strands.
  • Stabilize separated strands or Prevent re-annealing.
• Topoisomerase
  • Relieves torsional stress (supercoiling tension) ahead of replication fork.
  • Cuts and reseals DNA.
  • Prevents excessive supercoiling.
• Primase
  • Synthesizes short RNA primer.
  • Required because DNA polymerase cannot start de novo.
• DNA Polymerase
  • Extends primer by adding nucleotides.
  • Works strictly 5′ → 3′.
• DNA Ligase
  • Joins discontinuous fragments (Okazaki).
  • Forms phosphodiester bonds.

Transcription

• Definition
  • Transcription is the process of copying genetic information from one strand of DNA into RNA.
• Key Features
  • Only a specific segment of DNA is transcribed (not entire genome).
  • Only one of the two DNA strands is used as template.
  • Follows principle of base complementarity.
  • RNA is synthesized in 5′ → 3′ direction.
• Base Pairing Rule in Transcription
  • A pairs with U (instead of T)
  • G pairs with C

Why Only One Strand?

DNA is double-stranded, but both strands are NOT copied because:

Reason 1: Different Protein Problem

• If both strands acted as template:
  • Two different RNA molecules would be formed.
  • These RNAs would have different base sequences.
  • They would code for different proteins.
  • One DNA segment would generate two unrelated proteins.
• This would complicate genetic information flow.

Reason 2: Complementary RNA Problem

• The two RNA molecules formed would be complementary to each other:
  • They would bind together.
  • Form double-stranded RNA.
  • Double-stranded RNA cannot be translated.
• Thus, transcription would become useless.

Conclusion

• To maintain accuracy and simplicity of information transfer, only one strand acts as template.

Transcription Unit

A transcription unit is defined by three major regions on DNA:

1. Promoter:
   • Located at 5′ end of gene.
   • Provides binding site (starting point) for RNA polymerase.
   • Determines start site and direction of transcription.
   • Not transcribed.
2. Structural Gene:
   • DNA segment that is actually transcribed into RNA.
   • Contains information for RNA synthesis.
   • May be monocistronic or polycistronic.
     • Monocistronic → One gene → One polypeptide
     • Polycistronic → Multiple genes → Multiple polypeptides
3. Terminator:
   • Located at 3′ end of gene.
   • Signals termination of transcription.
   • Causes RNA polymerase to detach.
   • Thus acts as endpoint for transcription.

DNA Strands During Transcription

1. Template Strand:
   • Polarity: 3′ → 5′
   • Used by RNA polymerase.
   • mRNA formed is complementary to this strand.
2. Coding Strand:
   • Polarity: 5′ → 3′
   • Same sequence as mRNA.
   • Except T is replaced by U in mRNA.

Example

• DNA Template Strand: 3′-ATGCATGCATGC-5′
• DNA Coding Strand: 5′-TACGTACGTACG-3′
• Transcribed RNA would be: 5′-UACGUACGUACG-3′

Note- RNA sequence matches coding strand except T → U.

Genes and Transcription Units

Gene and Cistron

• Gene: Functional unit of inheritance located on DNA.
• Cistron: Segment of DNA coding for one polypeptide.
• Thus:
  • Monocistronic gene = one cistron or one gene per transcription unit.
  • Polycistronic gene = multiple cistrons or multiple genes per transcription unit.

Monocistronic gene mostly in eukaryotes, Polycistronic gene mostly in prokaryotes.

Gene Structure in Eukaryotes

Eukaryotic genes are discontinuous (split genes).

1. Exons:
   • Coding sequences that appear in mature RNA.
2. Introns:
   • Non-coding sequences removed during RNA processing and thus do not appear in mature RNA.

Regulatory Sequences:

• Affect inheritance and gene expression.
• Sometimes called regulatory genes, even though they don’t code for proteins or RNA.

Summary

• Transcription: Copying one DNA strand into RNA.
• Transcription Unit: Includes promoter, structural gene, and terminator.
• Genes: Functional units on DNA, can be monocistronic or polycistronic.
• Gene Structure: Includes exons and introns, regulated by promoter and other sequences.

Types of RNA and Transcription

Types of RNA:

• mRNA (Messenger RNA):
  • Act as the template for protein synthesis.
• tRNA (Transfer RNA):
  • Brings amino acids to ribosome and reads the genetic code.
  • Also called Adapter molecule.
• rRNA (Ribosomal RNA):
  • Structural + catalytic component of ribosome.
  • Forms ribosomal core.

Transcription in Prokaryotes (Bacteria)

• Occurs in cytoplasm.
• Requires RNA polymerase.

RNA Polymerase in Bacteria

• Consists of:
  • Core enzyme
  • Sigma factor (σ)
• Together called holoenzyme.
• Role of Sigma Factor:
  • Recognizes promoter sequences (-35 and -10 regions).
  • Ensures correct initiation site.
  • Provides specificity.

Steps of Transcription in Bacteria

1. Initiation:
   • RNA polymerase holoenzyme binds promoter region on DNA.
   • RNA polymerase starts transcription by opening the DNA helix.
   • Sigma recognizes -35 and -10 sequences.
   • DNA locally unwinds (~25 base pair region).
   • Transcription bubble forms.
   • After synthesis of first 8–9 nucleotides:
     • Sigma factor dissociates.
2. Elongation:
   • Core enzyme continues RNA synthesis.
   • RNA polymerase moves along template (3′ → 5′).
   • RNA synthesized in 5′ → 3′ direction.
   • Inside transcription bubble:
     • About 9 bases of RNA remain paired with DNA.
     • Bubble size ≈ 25 base pairs.
3. Termination:
   • Rho factor (ρ) binds to RNA.
   • Moves toward polymerase.
   • Reaches pause site.
   • Unwinds DNA–RNA hybrid.
   • RNA polymerase detaches.
   • RNA released.
   • Or Simply define Termination step like this:
     • When RNA polymerase reaches the terminator region, the new RNA and the enzyme (RNA polymerase) detach and new RNA strand is released.
     • This ends transcription.

RNA Polymerase Helpers

1. Initiation Factor (σ): Helps RNA polymerase start transcription.
2. Termination Factor (ρ): Helps RNA polymerase stop transcription.

Coupled Transcription and Translation in Bacteria (Imp. Concept)

• No nucleus in bacteria.
• Transcription and translation occur in same compartment (cytoplasm).
• Translation can begin even before transcription finishes.
• No RNA processing required.

This increases speed of protein synthesis in Bacteria.

Transcription in Eukaryotes

• More complex than prokaryotes.
• Occurs in nucleus.
• mRNA must be processed before translation.

RNA Polymerases in Eukaryotes

• Multiple RNA Polymerases:
  • RNA Polymerase I: Transcribes rRNA (28S, 18S, 5.8S).
  • RNA Polymerase III: Transcribes tRNA, 5S rRNA, and snRNAs.
  • RNA Polymerase II: Transcribes hnRNA (precursor to mRNA).
• hnRNA (Heterogeneous Nuclear RNA):
  • Primary transcript.
  • Also called pre-mRNA.
  • Longer than mature mRNA.
  • Contains both introns and exons.

Post-Transcriptional Modifications (RNA Processing)

Required only in eukaryotes.

1. Capping

• Addition of 7-methyl guanosine (7 mG).
• Added at 5′ end of hnRNA.
• Functions:
  • Protects mRNA from degradation.
  • Helps ribosome recognition.

2. Tailing

• Addition of poly-A tail (200–300 adenine residues).
• Added at 3′ end of hnRNA.
• Functions:
  • Increases stability.
  • Helps nuclear export.
  • Enhances translation efficiency.

3. Splicing

• Removal of introns (non-coding regions).
• Joining of exons (coding regions).
• Carried out by RNA splicing machinery {spliceosome (snRNP complex)}.
• Result:
  • Produces mature mRNA with continuous coding sequence (only exons).

Final Mature mRNA

• Contains only exons.
• Has 5′ cap.
• Has 3′ poly-A tail.
• Exported to cytoplasm.
• Ready for translation.

Significance of Complexities:

• The split-gene arrangement and presence of introns reflect ancient genome features.
• Splicing and RNA processing indicate the importance of RNA-based processes in living systems.

Summary

• Three Types of RNA: mRNA, tRNA, rRNA, each with different roles in protein synthesis.
• Transcription Steps: Initiation, Elongation, Termination.
• RNA Polymerase: Needs initiation and termination factors in bacteria.
• Eukaryotic Transcription: More complex, involves multiple RNA polymerases and processing steps for hnRNA.

Genetic Code

Concept of Genetic Code

• Genetic Code is the relationship between:
  1. Sequence of nucleotides in mRNA
  2. Sequence of amino acids in a polypeptide
• It is the dictionary that converts nucleotide language into amino acid language.
• Without genetic code, translation would not be possible.

Background (Central Dogma Context)

• Replication → DNA → DNA
• Transcription → DNA → RNA
• Translation → RNA → Protein

• There is NO direct chemical complementarity between: Nucleotides (DNA) and Amino acids.
• Therefore, a decoding system (genetic code) is required.

• Any change in DNA sequence
  • → Changes mRNA sequence
  • → May change amino acid sequence
  • → May change protein structure and function

Triplet Nature & Proposition of Genetic Code

• George Gamow proposed that:
  • There are 4 bases (A, U, C, G).
  • There are 20 amino acids.
  • A single base cannot code 20 amino acids.
  • A doublet code (4² = 16) is insufficient.
  • A triplet code (4³ = 64) is sufficient.
• Thus, three nucleotides form one codon.
• Total possible codons = 4 × 4 × 4 = 64

one codon = A sequence of three adjacent nucleotides in mRNA that codes for one amino acid

one codon = three nucleotides (a triplet) on the mRNA.

Deciphering the Genetic Code (Major Scientists Contribution)

• Marshall Nirenberg
  • Developed cell-free system for protein synthesis.
  • First codon discovered: UUU (codes for Phenylalanine).
• Har Gobind Khorana
  • Synthesized RNA with specific base sequences (homopolymers & copolymers).
• Severo Ochoa
  • Used polynucleotide phosphorylase to synthesize RNA.
• Robert Holley
  • Determined structure of tRNA.

Nobel Prize (1968): Holley, Nirenberg, Khorana.

Salient Features of Genetic Code

1. Triplet Code (nature):
   • Three nucleotides form one codon.
   • 61 codons code for amino acids.
   • 3 codons are stop codons.
2. Degeneracy:
   • Some amino acids are coded by more than one codon.
   • Or More than one codon can code for same amino acid.
   • Example: Phenylalanine → UUU and UUC.
   • Reason: Reduces harmful effect of mutations.
3. Non-overlapping:
   • Each nucleotide is read only once Or One base is part of only one codon.
   • Example: AUGUUU → AUG | UUU, (Not A UG UUU etc.)
4. Non-ambiguous:
   • One codon codes for only one amino acid.
   • Example: UUU always codes only for Phenylalanine.
5. Continuous (Comma-less):
   • Codons in mRNA are read continuously without punctuation.
   • Example: AUGUUUUUC, Read as AUG | UUU | UUC
6. Universality:
   • Same codon codes for the same amino acid in almost all organisms (e.g., UUU codes for Phenylalanine in bacteria as well as humans).
   • Thus most codons are universal across species.
   • Exceptions exist in mitochondrial codons and some protozoans.
7. Initiator Codon:
   • AUG Codes for Methionine & acts as an initiator codon.
8. Stop Codons (Nonsense Codons):
   • UAA (Ochre), UAG (Amber), UGA (Opal), signal the end of protein synthesis, thus these 3 act as stop codons.
9. Total: 61 codons for 20 amino acids & 3 codons are stop codons (Do not code for any amino acid).

• Note-
  • AUG and GUG are exceptions to the “non-ambiguous” rule.
  • In prokaryotes, GUG usually codes for Valine, but can act as a start codon for N-formylmethionine (fMet) if AUG is absent.

Wobble Hypothesis (Crick, 1965)

• Problem
  • If 61 codons exist, why not 61 tRNAs?
• Answer → Wobble Hypothesis
• Concept
  • First two bases of codon bind strictly with anticodon.
  • Third base binds loosely with anticodon.
  • Same tRNA can recognize multiple codons differing at third base..
• This explains degeneracy of code and, allows a single tRNA to recognize multiple codons for the same amino acid.
• Significance:
  • It reduces the number of tRNAs needed for translation, as one tRNA can read multiple codons, improving the efficiency of protein synthesis.

• Wobble Hypothesis = “wobble” position flexibility
• “wobble” position = It refers to the third nucleotide of an mRNA codon (and the first of a tRNA anticodon) where non-standard, flexible base pairing occurs, allowing one tRNA to recognize multiple codons.

Example Sequences

• mRNA Sequence: AUG UUU UUC UUC UUU UUU UUC
  • Amino Acid Sequence: Met-Phe-Phe-Phe-Phe-Phe-Phe. (Met =Methionine, Phe = Phenylalanine).

Mutations and Genetic Code

Mutation

• Mutations are changes in the DNA sequence.
• Large deletions and rearrangements can lead to the loss or gain of genes, affecting their functions.

1. Point Mutations

• Change (substitution) in single base pair.
• Example: Sickle Cell Anemia
  • Normal codon → GAG (Glutamic acid)
  • Mutated codon → GUG (Valine)
  • Result
    • Glutamate replaced by Valine
    • Hemoglobin structure altered
    • RBC become sickle-shaped

2. Frameshift Mutations

• Insertion or deletion of 1 or 2 bases.
• Shifts/Changes reading frame completely.
• Example with Sentence:
  • Original sentence: RAM HAS RED CAP
  • Insert B → RAM HAS BRE DCA P
  • Delete R → RAM HAS EDC AP
• Conclusion:
  • Insertion/deletion of 1 or 2 bases → Frameshift
  • Insertion/deletion of 3 bases → No frameshift

This proves codon is triplet and continuous.

Types of Mutations Based on Effect

• Missense Mutation
  • Codon changes → Different amino acid.
• Nonsense
  • Codon becomes stop codon → Premature termination.
• Silent
  • Codon changes → But same amino acid (due to degeneracy).

tRNA – The Adapter Molecule

• Proposed by Francis Crick.
• Function
  • Reads mRNA codon
  • Brings specific amino acid
  • Connects nucleotide language to amino acid language

Structure of tRNA

• Secondary Structure
  • Clover-leaf shape.
• Actual Structure
  • Compact, inverted L-shape.

Important Parts

1. Anticodon Loop
   • Complementary to mRNA codon.
2. Amino Acid Acceptor End
   • 3′ end (CCA sequence).
   • Binds specific amino acid.

Key Concepts

• Each tRNA is specific to one amino acid.
• Special initiator tRNA for AUG.
• No tRNA exists for stop codons.
• Stop codons are recognized by release factors.

Discovery: tRNA known before the genetic code was postulated.

Translation

• Definition:
  • Process of joining amino acids to form a polypeptide (a chain of amino acids).
  • “Translation is the process of polymerization of amino acids into a polypeptide.”
• Occurs in ribosome.
• Sequence determined by mRNA.
• Amino acids joined by peptide bonds.

Steps in Translation

Step 1: Activation of Amino Acids

• Amino acids activated using ATP (Enzyme used: Aminoacyl-tRNA synthetase).
• Amino acid attached to its specific tRNA (charging of tRNA or aminoacylation).

This step ensures accuracy.

Step 2: Formation of Peptide Bonds

• Energetically favored if charged tRNAs are close.
• Catalyzed by ribosome.

Role of Ribosome

Ribosome is the protein synthesis factory in a cell.

• Structure:
  • Made of structural RNAs and about 80 proteins.
  • Subunits: They consist of two different size subunits that lock around mRNA to catalyze peptide bond formation. .
    1. Small subunit (binds to mRNA to start translation).
    2. Large subunit
• Function: Synthesizes proteins by reading mRNA.
  • Catalytic Role: 23S rRNA in bacteria acts as an enzyme (ribozyme).

Thus, ribosome is ribozyme-based catalytic system.

Translation Process

Translational Unit in mRNA

• Translational Unit:
  • Part of mRNA that includes the start codon (AUG) and stop codon, coding for a polypeptide.
  • “Translational Unit = Sequence between start codon (AUG) and stop codon.”
• Untranslated Regions (UTR):
  • Parts of mRNA before the start codon and after the stop codon.
  • Not translated
  • Present at both 5′-end and 3′-end of mRNA.
  • They help in efficient translation & Stabilize mRNA.

Phases of Translation (General)

1. Initiation
   • Ribosome binds to mRNA at start codon (AUG).
   • Start codon is recognized by initiator tRNA.
2. Elongation
   • tRNA-amino acid complexes bind to codons in mRNA.
   • Ribosome moves along mRNA from codon to codon.
   • Amino acids added one by one.
3. Termination
   • A release factor binds to stop codon.
   • Translation ends, and polypeptide is released from ribosome.

Summary

• Translation: Converts genetic information in mRNA into a protein.
• Ribosome: Central role in protein synthesis.
• Process: Initiation, elongation, and termination.

Regulation of Gene Expression

What is Gene Expression?

• Definition:
  • Gene Expression is the process by which a gene produces a functional product, usually a polypeptide (protein).
• Regulation(controlling): Can occur at various levels to control how much polypeptide is made.
  • When a gene is expressed
  • How much protein is produced
  • Under what environmental or physiological conditions it is expressed

Why is Gene Expression Regulated?

• Cells do NOT produce all proteins all the time.
• Cells produce proteins only when needed.
• Reasons:
  • To conserve energy
  • To enable cell differentiation
  • To allow proper growth and development
  • To respond to environmental changes
• Example:
  • E. coli produces beta-galactosidase only when lactose is present.
  • If lactose is absent, enzyme production stops (To conserve energy).

Thus, metabolic, physiological, and environmental conditions regulate gene expression.

Types of Gene Regulation (Not mentioned in NCERT)

1. Negative Regulation
   • Gene expression is normally ON.
   • Repressor protein blocks/suppresses transcription.
   • When repressor binds → transcription stops.
   • Also called repressible regulation.
   • Example: Lac operon (primarily negative control).
2. Positive Regulation
   • Gene expression is normally OFF or weak.
   • Activator protein enhances transcription.
   • Activator helps RNA polymerase bind efficiently.
   • Also called inducible regulation.
   • Example: CAP-cAMP system in lac operon.

Levels of Regulation in Eukaryotes

Eukaryotic regulation is complex and can occur at multiple levels:

1. Transcriptional Level:
   • Control of primary RNA transcript formation.
   • Most important level.
2. Processing Level:
   • Regulation during splicing (introns removed, exons joined).
3. mRNA Transport:
   • Control of export of mRNA from nucleus to cytoplasm.
4. Translational Level:
   • Control of protein synthesis (from mRNA) efficiency/machinery.
5. Post-translational Level (advanced concept): “Not mentioned in NCERT”
   • Protein modification after synthesis (phosphorylation, glycosylation etc.).

Regulation in Prokaryotes

In prokaryotes, regulation mainly occurs at: Transcription Initiation

• Reason:
  • No nucleus
  • Transcription and translation are coupled
  • Rapid response needed
• RNA polymerase binding to promoter is regulated by regulatory proteins.

Regulatory Proteins

• Repressors
  • Bind operator region
  • Block RNA polymerase
  • Decrease transcription
• Activators
  • Bind near promoter
  • Help RNA polymerase bind
  • Increase transcription

Operon Concept

• Operon:
  • A group of genes regulated together.
  • A functional unit of DNA.
  • Common in bacteria.
  • Usually polycistronic.
• Example:
  • Lac operon in E. coli.
  • other examples include trp operon, ara operon, his operon, val operon, etc.

Components of an Operon

1. Promoter
   • Binding site for RNA polymerase to start transcription.
2. Operator
   • Binding site for repressor protein.
3. Structural Genes
   • Code for enzymes or proteins involved in related functions.

Each operon has its own specific operator and repressor.

Summary

• Gene Expression: Controlled at multiple levels.
• Prokaryotes: Mainly regulated at transcription initiation.
• Operons: Specific to each set of genes, like the lac operon in E. coli.

The Lac Operon

What is the Lac Operon?

• Discovered by:
  • Francois Jacob and Jacques Monod.
• Lac = Lactose.
• It is a transcriptionally regulated system controlling lactose metabolism.

Components of Lac Operon

1. One Regulatory Gene
   • i gene
2. Three Structural Genes
   • z, y, a

Regulatory Gene (i gene)

• Codes for lac repressor protein.
• Repressor is synthesized continuously (constitutive expression).

Structural Genes

1. z gene
   • Codes for beta-galactosidase
   • Breaks lactose into glucose and galactose.
2. y gene
   • Codes for permease
   • Allows lactose entry into cell.
3. a gene
   • Codes for transacetylase
   • Assists in lactose metabolism.

Working of Lac Operon

Case 1: Absence of Lactose

• Steps:
  1. Repressor protein is produced by i gene.
  2. Repressor binds to operator region.
  3. RNA polymerase blocked.
  4. No transcription of structural genes.
  5. No enzyme production.

Operon is OFF.

Case 2: Presence of Lactose

Lactose acts as Inducer. {However, Actual inducer is allolactose (converted form of lactose).}

• Steps:
  1. Lactose enters cell via permease.
  2. Lactose converts into allolactose.
  3. Allolactose binds to repressor.
  4. Repressor changes shape (conformational change) & becomes inactive.
  5. Inactive repressor cannot bind operator.
  6. RNA polymerase binds promoter.
  7. Structural genes are transcribed.
  8. Enzymes for lactose metabolism are produced.

Operon is ON.

Important Points

• Lactose is the inducer.
• Allolactose actually inactivates the repressor.
• Glucose is NOT an inducer.
• Lac operon is an example of negative regulation.

Why Lac Operon is Efficient

• Enzymes produced only when lactose present.
• Strong expression only when glucose absent.
• Saves energy.
• Shows environmental adaptation.

Comparison: Prokaryotic vs Eukaryotic Regulation

1. Prokaryotes
   • Mainly transcription-level regulation
   • Operon system present
   • Polycistronic mRNA
   • Rapid response
2. Eukaryotes
   • Multi-level regulation
   • No operon system
   • Mostly monocistronic mRNA
   • Complex control mechanisms

Summary

• Gene expression is regulated to conserve energy and ensure proper function.
• Prokaryotes regulate mainly at transcription initiation.
  • Negative regulation → Repressor blocks transcription.
  • Positive regulation → Activator enhances transcription.
• Lac operon → example of negative regulation.
• Lac operon regulates lactose metabolism.
• Operon system, groups related genes.
• Lactose acts as inducer.
• Repressor protein enables negative regulation.
• Eukaryotes regulate at multiple levels.

Human Genome Project

What is the Human Genome Project (HGP)?

• Purpose: The Human Genome Project (HGP) was a large international scientific research program aimed at:
  • Determining the complete nucleotide sequence of the human genome
  • Identifying all human genes
  • Understanding their structure and potential functions

• Launch Year: 1990
• Completion: 2003 (13-year project).

It is also called the International Human Genome Sequencing Consortium.

Coordinating Agencies

• U.S. Department of Energy (DOE)
• National Institutes of Health (NIH)
• Major partner: Wellcome Trust (UK)
• Other contributors: Japan, France, Germany, China and others

The project led to rapid development of bioinformatics as a discipline.

Why Sequence the Human Genome?

• Genetic information is stored in DNA sequence.
• Differences among individuals arise due to differences in DNA sequence.
• Understanding the sequence helps in:
  • Disease diagnosis
  • Identifying disease genes
  • Drug development
  • Understanding evolution

Major Goals of HGP

1. Identify Genes: Find all the 20,000-25,000 genes in human DNA.
2. Sequence DNA: Determine the sequences of the 3 billion base pairs in human DNA.
3. Store Information: Store DNA sequence information in databases.
4. Data Analysis: Improve bioinformatics tools for analyzing this data.
5. Technology Transfer: Share related technologies with other industries.
6. Address Issues: Address Ethical, Legal and Social Issues (ELSI)

ELSI was an important part of HGP because genetic data affects privacy and medical ethics.

Scale of the Project

• Human genome: Human genome has about 3 billion base pairs.
• Estimated cost: Initially estimated at $3 per base pair, totaling about 9 billion US dollars.
• Data size: ~3300 books (1000 pages each) to store the DNA sequence from a single human cell.

Methodologies Used in HGP

1. Expressed Sequence Tags (ESTs)
   • Focused on sequencing only expressed genes (RNA transcripts).
   • Helped identify active genes.
2. Whole Genome Shotgun Sequencing
   • Sequencing entire genome including coding and non-coding regions.

Sequencing Process

1. DNA Isolation and Fragmentation:
   • DNA isolated from cells.
   • Broken into smaller fragments for sequencing.
2. Cloning of Fragments:
   • Amplifying DNA fragments using host like bacteria or yeast with BACs (bacterial artificial chromosomes) and YACs (yeast artificial chromosomes).
3. DNA Sequencing:
   • Automated DNA sequencers used.
   • Based on Sanger’s chain termination method.
4. Alignment and Annotation:
   • Overlapping fragments are aligned using computer programs to assemble the full sequence and also assign (their positions) them to chromosomes.
   • Identifying genes and functional regions also.

Important Milestones

• Chromosome 1: Chromosome 1 was the last human chromosome sequenced (2006).
• Genetic and Physical Maps: Physical and genetic maps created using DNA polymorphisms, important for DNA fingerprinting.

Salient Features of the Human Genome

1. Genome Size and Content:
   • Genome Size: 3164.7 million base pairs
   • Gene Number: ~30,000 genes (much lower than earlier estimate of 80,000–140,000)
     • Largest Gene: Dystrophin gene (~2.4 million bases)
   • Average Gene Length: ~3000 bases
2. Genetic Similarity:
   • Almost 99.9% DNA identical among all humans
3. Unknown Functions:
   • Over 50% genes have unknown functions
4. Protein-Coding Genes:
   • Less than 2% of the genome codes for proteins.
5. Repeated Sequences:
   • A large portion of the genome is made up of repeated sequences.
   • These repetitive sequences don’t code for proteins but are important for understanding chromosome structure, dynamics, and evolution.
6. Chromosome Distribution:
   • Chromosome 1 → highest number of genes (2968)
   • Y chromosome → fewest genes (231)
7. Single Nucleotide Polymorphisms (SNPs):
   • About 1.4 million locations of single-base DNA differences, called SNPs, have been identified.
   • SNPs useful for disease gene mapping and evolutionary studies

Applications and Future Challenges

• Medical Applications:
  • Identification of disease-causing genes
  • Early diagnosis
  • Pharmacogenomics (personalized medicine)
• Research Applications:
  • Functional genomics
  • Systems biology
  • Studying gene networks
• Other Fields:
  • Agriculture
  • Biotechnology
  • Evolutionary biology
  • Future Research

Future Challenges

• Understanding function of unknown genes
• Studying gene–gene interactions
• Studying gene–environment interactions
• Large-scale systems biology approaches

DNA Fingerprinting

What is DNA Fingerprinting?

• Definition & Purpose: DNA fingerprinting is a technique used to identify individuals based on variations in specific DNA regions.
  • Although 99.9% of human DNA is identical,
  • the remaining 0.1% variation makes each individual unique (except identical twins).

Basis of DNA Fingerprinting

• The technique is based on: Repetitive DNA Regions
  • Short DNA sequences repeated many times in tandem
  • Highly polymorphic
  • Called Variable Number of Tandem Repeats (VNTRs)

Satellite DNA

A type of repetitive DNA that consists of long tandem repeats and is typically found in centromeric and heterochromatic regions of chromosomes.

• Separation: During density gradient centrifugation:
  • Bulk genomic DNA forms major peak
  • Repetitive DNA forms minor peaks
  • These are called satellite DNA
• Types of Satellite DNA:
  1. Minisatellites
  2. Microsatellites
• Location:
  • Mostly in centromeric and heterochromatic regions
• Role:
  • Though they don’t code for proteins, they help in understanding chromosome structure and evolution.

Polymorphism

• Definition: Variations in DNA sequences at a locus (Presence of more than one allele at a locus) in a population (frequency >1%).
  • Arises due to mutation
  • More common in non-coding regions
  • Inherited from parents
  • Basis of forensic identification

Technique of DNA Fingerprinting

• Developer: Sir Alec Jeffreys

Classical Technique (Southern Blot Method)

Probe Used: Variable Number of Tandem (sequential) Repeats (VNTR).

• Step 1: DNA Isolation
  • From blood, hair follicle, semen, saliva etc.
• Step 2: DNA Digestion
  • Cut the DNA into fragments using restriction enzymes
• Step 3: Gel Electrophoresis
  • Separate DNA fragments based on size.
• Step 4: Denaturation
  • Double-stranded DNA converted to single-stranded
• Step 5: Southern Blotting
  • Blot (Transfer) the separated DNA onto a synthetic membrane (nylon membrane).
• Step 6: Hybridization
  • Radiolabeled VNTR probe added
  • Probe (tag) binds to specific DNA sequences.
• Step 7: Autoradiography
  • X-ray film reveals band pattern
  • Each individual produces a unique banding pattern.

Characteristics of VNTR

• Meaning: A type of satellite DNA used in early DNA fingerprinting.
• Structure: Tandemly (sequential) repeated small DNA sequences.
• Polymorphism: High degree of variations; size varies from 0.1 to 20 kb.
• Pattern: Creates unique band patterns for each individuals (except identical twins have identical patterns).

Modern Improvement: PCR-Based Fingerprinting (Not mentioned in NCERT)

• PCR (Polymerase Chain Reaction)
  • Amplifies very small DNA samples
  • Allows analysis from a single cell
  • Faster and more sensitive
• Today, STR (Short Tandem Repeat) analysis is commonly used.

Applications of DNA Fingerprinting

• Forensics: Identification in crime scenes.
  • Crime Scene Investigation: DNA from a crime scene is compared with suspects’ DNA.
  • A match in the banding pattern identifies the suspect.
• Paternity Testing: Determine parentage by comparing DNA sequences.
• Genetic Mapping: Study genetic differences in populations.
• Genetics: Studying genetic diversity and population genetics.
• Sensitivity: Enhanced by PCR, allowing analysis from a single cell.

Important Terms

• VNTR (Variable Number of Tandem Repeats): A type of satellite DNA used in early DNA fingerprinting.
• Polymorphism: Genetic variations that occur in more than 1% of the population.
• PCR (Polymerase Chain Reaction): A technique to amplify small DNA samples, enhancing the sensitivity of DNA fingerprinting.

Quick Comparison

• Human Genome Project
  • Sequencing entire genome
  • Large-scale global research project
  • Focused on mapping and understanding genome
• DNA Fingerprinting
  • Identifies individuals
  • Based on VNTR polymorphism
  • Applied in forensic and legal science

Chapter Summary

NUCLEIC ACIDS

• Nucleic acids are polymers made of nucleotides.
• Each nucleotide contains:
  1. Nitrogenous base
  2. Pentose sugar
  3. Phosphate group

DNA vs RNA

• DNA
  • Genetic material in most organisms
  • Stores hereditary information
• RNA
  • Acts as messenger, adapter and catalytic molecule
  • Genetic material in some viruses
• Stability Difference
• DNA is more stable because:
  • Deoxyribose lacks 2′-OH group
  • Double-stranded structure
  • Thymine replaces uracil
  • Has repair mechanisms

RNA World Concept

• RNA was the first genetic material
• Functioned as both genetic material and catalyst
• DNA evolved later for greater stability

STRUCTURE OF DNA

• Double Helix Model (Watson & Crick, 1953)
  • Two antiparallel polynucleotide strands
  • Sugar-phosphate backbone outside
  • Bases project inward
• Base Pairing (Complementarity)
  • A = T (2 hydrogen bonds)
  • G = C (3 hydrogen bonds)
  • Purine always pairs with pyrimidine
• Chargaff’s Rule
  • A = T
  • G = C
  • A + G = T + C
• Helical Features
  • Right-handed helix
  • 10 base pairs per turn
  • Pitch = 3.4 nm
  • Distance between base pairs = 0.34 nm
• Stability
  • Hydrogen bonds
  • Base stacking interactions

DNA REPLICATION

• Mode of Replication: Semiconservative
• Each daughter DNA contains:
  • – One parental strand
  • – One newly synthesized strand
• Proven By
  • Meselson and Stahl (1958) – E. coli (15N experiment)
  • Taylor et al. (1958) – Vicia faba

• Key Enzyme
  • DNA-dependent DNA polymerase
• Important Features
  • Works only in 5′ → 3′ direction
  • Leading strand → continuous
  • Lagging strand → discontinuous (Okazaki fragments)
  • Occurs at replication fork
  • Initiates at specific origin (Ori)
  • Occurs in S-phase (eukaryotes)

GENE AND TRANSCRIPTION

• Gene
  • A segment of DNA that codes for a functional RNA molecule.
• Transcription
  • Copying genetic information from DNA to RNA
  • Only one DNA strand is transcribed
  • RNA synthesized in 5′ → 3′ direction
  • A pairs with U during transcription

• Transcription Unit
  1. Promoter
  2. Structural gene
  3. Terminator

• Prokaryotes
  • mRNA is directly functional
  • Transcription and translation are coupled
  • Polycistronic mRNA common

• Eukaryotes
  • Genes contain exons and introns
  • hnRNA (pre-mRNA) formed
  • RNA processing required:
    1. Capping
    2. Tailing
    3. Splicing

GENETIC CODE

• Definition
  • Genetic code is the relationship between codons in mRNA and amino acids in proteins.
• Triplet Nature
  • Three nucleotides form one codon
  • 64 total codons
  • 61 sense codons
  • 3 stop codons

• Salient Features
  • Degenerate (multiple codons for same amino acid)
  • Non-overlapping
  • Non-ambiguous
  • Comma-less (continuous)
  • Nearly universal

• Start Codon
  • AUG → Methionine
• Stop Codons
  • UAA, UAG, UGA
• Wobble Hypothesis
  • Third base pairing is flexible
  • Explains degeneracy

TRANSLATION

• Definition
  • Process of polymerization of amino acids to form a polypeptide.

Components

1. mRNA
   • Carries codon sequence
2. tRNA (Adapter Molecule)
   • Has anticodon
   • Has amino acid acceptor arm
   • Specific for each amino acid
3. Ribosome
   • Site of protein synthesis
   • Consists of rRNA and proteins
   • rRNA acts as ribozyme (peptide bond formation)

Stages

1. Initiation
2. Elongation
3. Termination

Energy Requirement

• ATP and GTP required
• Highly energy-intensive process

REGULATION OF GENE EXPRESSION

• Purpose
  • Cells synthesize proteins only when required.
• Primary Regulation Site
  • Mainly at transcription level (especially in prokaryotes)

Operon Concept (Prokaryotes)

• Operon = Cluster of genes regulated together

Components

• Promoter
• Operator
• Structural genes
• Regulatory gene

Lac Operon

• Controls lactose metabolism
• Lactose (allolactose) acts as inducer
• Absence of lactose → repressor blocks transcription
• Presence of lactose → repressor inactivated → operon ON
• Example of negative regulation

Eukaryotes

• Regulation occurs at multiple levels:
  • Transcriptional
  • Post-transcriptional
  • Translational
  • Post-translational

HUMAN GENOME PROJECT (HGP)

• Aim
  • Sequence entire human genome
  • Identify all human genes
• Launched: 1990
• Completed: 2003

• Key Findings
  • Genome size ≈ 3.16 billion base pairs
  • ~30,000 genes
  • <2% codes for proteins
  • 99.9% DNA identical among humans
  • ~1.4 million SNPs identified
• Significance
  • Disease gene identification
  • Pharmacogenomics
  • Foundation of genomics & bioinformatics

DNA FINGERPRINTING

• Principle
  • Based on DNA polymorphism
  • Uses Variable Number of Tandem Repeats (VNTR)
• Polymorphism
  • Variation at genetic level
  • Frequency >1% in population
• Developer
  • Alec Jeffreys
• Applications
  • Forensic identification
  • Paternity testing
  • Evolutionary studies
  • Population genetics
• Modern Method
  • PCR-based STR analysis