The DNA
Structure of Polynucleotide Chain
- Nucleotide components: A nitrogenous base, a pentose sugar (deoxyribose in DNA, ribose in RNA), and a phosphate group
- Nitrogenous bases:
- Purines: Adenine and Guanine (found in both DNA and RNA) NEET 2019
- Pyrimidines: Cytosine (common to both), Thymine (exclusive to DNA), Uracil (exclusive to RNA)
Linkages in DNA
- N-glycosidic linkage: Joins nitrogenous base to 1'C-OH of pentose sugar → forms nucleoside
- Phosphoester linkage: Joins phosphate group to 5'C-OH of nucleoside → forms nucleotide
- 3'-5' phosphodiester linkage: Joins two consecutive nucleotides → forms dinucleotide/polynucleotide 2010
Polymer ends: 5' end has free phosphate moiety; 3' end has free 3'-OH group
DNA Dimensions
Length defined by base pairs (bp):
- φ×174 bacteriophage: 5386 nucleotides 2022 Re
- Lambda bacteriophage: 48502 bp
- E. coli: 4.6×10⁶ bp
- Human haploid DNA: 3.3×10⁹ bp
Nuclein: Acidic substance in nucleus first identified by Friedrich Meischer (1869)
DNA Double Helix Model
Proposed by James Watson and Francis Crick (1953) based on X-ray diffraction by Maurice Wilkins and Rosalind Franklin
Erwin Chargaff's Rule
For double-stranded DNA, ratios between Adenine/Thymine and Guanine/Cytosine are constant and equal one 2015
Salient Features
- Two polynucleotide chains with sugar-phosphate backbone; bases project inside
- Anti-parallel polarity: One strand is 5'→3', the other is 3'→5' 2008, 2006
- Base pairing: Adenine pairs with Thymine via two H-bonds NEET 2020. Guanine pairs with Cytosine via three H-bonds. Purine always opposite pyrimidine
- Right-handed coiling: Pitch = 3.4 nm. 10 bp per turn. Distance between bp = 0.34 nm 2006, 2000
- Plane of one base pair stacks over the other, conferring stability to helical structure
Central Dogma
Proposed by Francis Crick: DNA → mRNA → Protein NEET 2013, 2021
Exception/Crucial detail: Retroviruses (e.g., HIV) exhibit reverse flow (RNA → DNA) using Reverse Transcriptase 2010, 2005
Packaging of DNA Helix
Prokaryotes (e.g., E. coli)
- Lack defined nucleus. DNA (negatively charged) held in large loops by proteins (positively charged) in region called nucleoid NEET 2023
Eukaryotes
- Complex organisation involving basic, positively charged proteins called histones 1997
- Histones rich in basic amino acid residues lysine and arginine 2021, 2022
- Organized to form a histone octamer 1990
- Nucleosome: Negatively charged DNA wrapped around positively charged histone octamer NEET 2023. Typical nucleosome contains 200 bp of DNA helix 2022 Re
- Chromatin: Repeating nucleosome units form thread-like bodies, seen as 'beads-on-string' under electron microscope 2011
Exception/Crucial detail: Association of H1 histone with nucleosome indicates DNA is condensed into a chromatin fibre NEET 2017
Higher-level Packaging
Chromatin coils into chromosomes at metaphase, requiring Non-histone Chromosomal (NHC) proteins
| Chromatin Type | Packaging | Staining | Transcriptional Activity |
|---|---|---|---|
| Euchromatin | Loosely packed 2022, 2024 Re | Light | Active |
| Heterochromatin | Densely packed 2022, 2024 Re | Dark | Inactive |
The Search for Genetic Material
Transforming Principle
Frederick Griffith (1928): Conducted experiments on Streptococcus pneumoniae 2002, 2014
- S strain: Smooth mucous (polysaccharide) coat, virulent (kills mice)
- R strain: Rough, non-virulent
- Experiment: Heat-killed S strain + live R strain injected → mice died. Live S bacteria recovered
- Conclusion: R strain transformed by a 'transforming principle' from heat-killed S strain
Biochemical Characterisation
Avery, MacLeod, and McCarty (1933-44) purified proteins, DNA, RNA from heat-killed S cells
- Proteases and RNases did NOT affect transformation
- DNase inhibited transformation, proving DNA is the hereditary material 1993
The Genetic Material is DNA
Unequivocal Proof: Provided by Alfred Hershey and Martha Chase (1952) using bacteriophages (T₂ virus) infecting E. coli 1993, 2023, 2023 Manipur
Experiment Mechanism
- Grew viruses on radioactive phosphorus (³²P) → labelled DNA
- Grew viruses on radioactive sulfur (³⁵S) → labelled protein
- Infection → Blending (removes viral coats) → Centrifugation
- Result: Radioactive phosphorus (³²P) detected inside bacterial cells; Radioactive sulfur (³⁵S) detected in supernatant 2023 Manipur. Proved DNA passed from virus to bacteria
RNA World & Properties of Genetic Material
Criteria for Genetic Material
Must generate replica, be chemically/structurally stable, provide scope for slow mutations, and express Mendelian characters NEET-II 2016
DNA vs RNA Comparison
| Property | DNA | RNA |
|---|---|---|
| Stability | Chemically less reactive, structurally more stable (Thymine provides stability; lacks highly reactive 2'-OH group) | Less stable due to reactive 2'-OH group |
| Mutation Rate | Slower mutation rate | Unstable; mutates faster. RNA viruses have shorter life spans and evolve faster 2023 Manipur |
| Expression | Depends on RNA for protein synthesis | Directly codes for proteins |
RNA World: RNA was the first genetic material. Essential life processes (metabolism, translation, splicing) evolved around RNA. RNA acts as a catalyst (ribozyme) 2016 but is unstable. DNA evolved from RNA with chemical modifications for stability
Replication
Semiconservative DNA Replication
Proposed by Watson and Crick. Each parental strand acts as a template for a new complementary strand
Experimental Proof (Meselson & Stahl, 1958)
Performed on E. coli 2018
- Grew E. coli in heavy nitrogen (¹⁵NH₄Cl) for many generations
- Transferred to normal nitrogen (¹⁴NH₄Cl) and centrifuged in CsCl density gradient
- Generation 1 (20 mins): Hybrid density (¹⁵N/¹⁴N)
- Generation 2 (40 mins): Equal amounts of hybrid and light (¹⁴N/¹⁴N) DNA
Taylor et al. (1958): Proved semiconservative replication in chromosomes using radioactive thymidine on Vicia faba (faba beans) NEET-II 2016
Machinery and Enzymes
- DNA-dependent DNA polymerase: Main enzyme. Highly efficient, accurate, but catalyses polymerisation only in one direction: 5'→3' 2022 Re, 2024
- Deoxyribonucleoside triphosphates (dNTPs): Serve dual purpose: act as substrates AND provide energy for polymerisation
Replication Fork Mechanisms
- Continuous synthesis (Leading strand): Occurs on template strand with polarity 3'→5'
- Discontinuous synthesis (Lagging strand): Occurs on template strand with polarity 5'→3'. Fragments (Okazaki fragments) elongate lagging strand away from replication fork NEET 2017
- DNA ligase: Joins discontinuously synthesised fragments 1994
- Helicase: Facilitates opening of DNA helix 1993, 2020
- Origin of replication (ori): Definite region where replication initiates
Transcription
Transcription Unit
Definition: Copying genetic info from one DNA strand into RNA. Only one strand is copied to avoid forming dsRNA and producing differing proteins
Components
A Promoter, Structural gene, and Terminator 2012
Template vs Coding Strand
NEET-II 2016, 2014
- Template strand: Polarity 3'→5'. Directs RNA synthesis
- Coding strand: Polarity 5'→3'. Has same sequence as newly synthesised RNA (except T instead of U). All reference points (upstream/downstream) made relative to this strand
Promoter: Located at 5' end (upstream) of coding strand. Binding site for DNA-dependent RNA polymerase 2003
Process of Transcription
Bacteria (Prokaryotes)
- Single DNA-dependent RNA polymerase transcribes all types of RNA (mRNA, tRNA, rRNA)
- Initiation: RNA polymerase binds promoter, transiently associates with initiation factor (σ)
- Elongation: RNA polymerase directly catalyses elongation 2024 Re, 2021 using nucleoside triphosphates
- Termination: Polymerase reaches terminator, associates transiently with termination factor (ρ), releasing nascent RNA 2024 Re, 2021
Note: Transcription and translation can be coupled in bacteria
Eukaryotes
Three different RNA polymerases with clear division of labour 2024 Re
| Eukaryotic Enzyme | Transcribed Products | High-Yield Notes |
|---|---|---|
| RNA Polymerase I | rRNAs (28S, 18S, 5.8S) | Active in the nucleolus 2012 |
| RNA Polymerase II | hnRNA (mRNA precursor) | Transcribes structural genes |
| RNA Polymerase III | tRNA, 5S rRNA, snRNAs | Transcribes adapter & small nuclear RNAs 2023, 2024 Re |
Post-Transcriptional Processing (Eukaryotes)
- Structural genes are split into Exons (coding/expressed sequences) and Introns (intervening/non-coding sequences)
- Splicing: Removal of introns and joining of exons in defined order 2012. Represents dominance of RNA world. Spliceosomes are absent in bacteria NEET 2017
- Capping: Addition of methyl guanosine triphosphate to 5' end of hnRNA 2021
- Tailing (Polyadenylation): Addition of 200-300 adenylate residues at 3' end. Fully processed hnRNA is now mRNA
Genetic Code
Features of the Genetic Code
- Triplet: 61 codons code for 20 amino acids; 3 act as stop codons 1990, 2003
- Unambiguous & Specific: One codon codes for only one amino acid
- Degenerate: Some amino acids are coded by more than one codon 2010
- Contiguous: Read in mRNA without punctuations
- Nearly Universal: From bacteria to humans, UUU codes for Phenylalanine. This feature allows recombinant DNA technology to produce human insulin in bacteria NEET 2019. Exceptions exist in mitochondrial codons and some protozoans
- Dual Function of AUG: Acts as Initiator codon AND codes for Methionine 1996, 2016
- Stop Codons: UAA, UAG, UGA terminate translation 1996, 1997
Mutations & Genetic Code
- Point Mutation: Change in single base pair. Example: Sickle cell anemia (glutamate to valine in beta-globin chain) 2023 Manipur
- Frameshift Mutation: Insertion or deletion of one/two bases shifts reading frame. Insertion/deletion of three multiples keeps reading frame intact but adds/removes amino acids
tRNA - The Adapter Molecule
- Reads genetic code and binds to specific amino acids
- Structure: Has an anticodon loop complementary to mRNA codon 2000 and an amino acid acceptor end (3') 1995. Looks like clover-leaf (secondary structure) but actually compact inverted L 2004
- Initiator tRNA exists for start codon; NO tRNAs exist for stop codons
Translation
Definition: Polymerisation of amino acids to form a polypeptide
Charging of tRNA (Aminoacylation)
Amino acids activated in presence of ATP and linked to their cognate tRNA. Consumes high-energy phosphate bond 1997
Ribosome
- Cellular factory for protein synthesis. Consists of structural RNAs and ~80 proteins 2023
- Small subunit encounters mRNA → translation begins 2022
- Large subunit has two sites for subsequent amino acids to bind close enough for peptide bond formation
- Ribozyme: 23S rRNA in bacteria acts as catalyst for peptide bond formation 2016
Polysome (Polyribosome)
Multiple ribosomes attached to single mRNA strand to simultaneously translate multiple copies of polypeptide NEET 2018, 2016
UTRs (Untranslated Regions)
Present at both 5' (before start) and 3' (after stop) ends. Required for efficient translation
Termination
A release factor binds to stop codon, terminating translation
Regulation of Gene Expression
Overview
Gene expression can be regulated at multiple levels in eukaryotes: Transcriptional, Processing (splicing), Transport of mRNA to cytoplasm, and Translational
In prokaryotes, transcriptional initiation is predominant site for control
The Lac Operon
Elucidated by Francois Jacob and Jacque Monod 2018. A polycistronic structural gene regulated by common promoter and regulatory genes
Components
- i gene: Regulatory gene. Synthesises repressor protein constitutively (all the time) 2022 Re
- Promoter (p): Binding site for RNA polymerase
- Operator (o): Binding site for repressor protein
- z gene: Codes for β-galactosidase (hydrolyses lactose into galactose and glucose) 2024 Re
- y gene: Codes for permease (increases cell permeability to β-galactosides)
- a gene: Codes for transacetylase
Mechanism
- Absence of Inducer: Repressor binds to operator, preventing RNA polymerase from transcribing operon (Negative regulation) 2015
- Presence of Inducer: Lactose or allolactose acts as inducer 2023 Manipur, 1994. It binds and inactivates repressor, allowing transcription to proceed
Exception/Crucial detail: Glucose or galactose CANNOT act as inducers for lac operon 2010, 2024 Re
Human Genome Project (HGP)
A 13-year mega project (1990-2003) to sequence human genome (3×10⁹ bp). Spurred development of Bioinformatics
Methodologies
- Expressed Sequence Tags (ESTs): Identifying all genes expressed as RNA NEET 2019, 2023
- Sequence Annotation: Blind sequencing of entire genome (coding and non-coding), later assigning functions to regions 2022
- Tools: BAC and YAC vectors. Automated sequencers based on Frederick Sanger's method
Salient Features of Human Genome
- Contains 3164.7 million bp. Total genes estimated at ~30,000
- Average gene = 3000 bases; Largest gene = Dystrophin (2.4 million bases)
- 99.9% nucleotide bases are exactly same in all humans
- < 2% of genome codes for proteins
- Repeated sequences make up very large portion
- Chromosome 1 has most genes (2968); Chromosome Y has fewest (231). Chromosome 1 was last to be sequenced (May 2006) 2023 Manipur
- 1.4 million locations possess SNPs (Single Nucleotide Polymorphisms)
DNA Fingerprinting
Developed by Alec Jeffreys. Technique to compare DNA sequences of individuals quickly
Basis
Identifies differences in repetitive DNA sequences, which show high degrees of polymorphism (variation at genetic level) 2022, 2015
Satellite DNA
Small peaks in density gradient centrifugation. Classified into micro/mini-satellites based on base composition, length, and copy number. Does not code for proteins
VNTR (Variable Number of Tandem Repeats)
A mini-satellite used as radiolabelled probe. Shows high polymorphism; varies in size from 0.1 to 20 kb
Steps in DNA Fingerprinting
- Isolation of DNA
- Digestion by restriction endonucleases
- Separation by electrophoresis
- Transferring (blotting) to synthetic membranes (nitrocellulose/nylon)
- Hybridisation using labelled VNTR probe
- Detection by autoradiography
Note: Techniques do NOT require Zinc finger analysis NEET-I 2016. PCR increases sensitivity