Biotechnology: Class 11 — Chapter 7: Basic Processes

In 1928, Frederick Griffith demonstrated genetic transformation using Streptococcus pneumoniae in mice:

• S-strain (Smooth): Virulent; possesses a protective polysaccharide capsule that prevents detection by the host immune system, causing lethal pneumonia.
• R-strain (Rough): Non-virulent; lacks the polysaccharide capsule and is easily cleared by the host immune system.
• Transformation Event: Injection of heat-killed S-strain combined with live R-strain resulted in mouse death. Viable, live S-strain bacteria were recovered from the blood, proving that a chemical component from the dead S-strain transformed the live R-strain into a virulent phenotype.

In 1944, Oswald Avery, Colin Macleod, and Maclyn McCarty purified biochemical fractions (proteins, RNA, DNA) from heat-killed S-strain bacteria to identify the transforming agent:

• Treatment of the active transforming fraction with Protease (protein-degrading) or RNase (RNA-degrading) enzymes did not inhibit transformation.
• Treatment with DNase (DNA-degrading enzyme) completely abolished transforming activity.
• Conclusion: DNA is the chemical substance responsible for transformation and is the genetic material.

In 1952, Alfred Hershey and Martha Chase provided definitive proof using bacteriophage T2 and Escherichia coli:

• Isotopic Labeling: Phage proteins were selectively labeled with radioactive sulfur ($^{35}\text{S}$) in their disulfide bonds, while phage DNA was labeled with radioactive phosphorus ($^{32}\text{P}$) in its sugar-phosphate backbone.
• Experimental Workflow: Labeled phages were allowed to infect unlabelled E. coli cells. The mixture was agitated in a blender to shear off extracellular phage coats, followed by centrifugation to separate bacterial cells (pellet) from viral debris (supernatant).
• Analytical Results: Radioactivity from $^{32}\text{P}$ was detected exclusively inside the bacterial pellet, whereas $^{35}\text{S}$ remained in the supernatant. This confirmed that viral DNA, not protein, enters the host cell to direct viral replication.

• Nucleoid Region: Prokaryotes lack a nuclear membrane; their circular, double-stranded DNA is localized in a specialized cytoplasmic region.
• Supercoiling: To fit within the cell, circular DNA undergoes twisting catalyzed by topoisomerases. Genomes can be negatively supercoiled (twisted opposite to the double helix direction) or positively supercoiled. Most bacterial genomes are negatively supercoiled during active growth.
• Plasmids: Small, circular, self-replicating extrachromosomal DNA molecules that carry non-essential but advantageous genes (e.g., antibiotic resistance).
• HLU (Histone-like Proteins): Abundant basic proteins in the nucleoid that assist in DNA condensation.

Eukaryotic DNA is linear, highly elongated, and packaged into a complex nucleoprotein structure called chromatin:

• Histones: Highly basic proteins rich in positively charged amino acids (arginine and lysine) that bind tightly to negatively charged DNA phosphate groups.
• Nucleosome Core: Composed of an octamer containing two molecules each of histones H2A, H2B, H3, and H4. Approximately 146 base pairs (bp) of DNA wrap $1.65$ times around this octamer core.
• Linker DNA: A $54$ bp segment of DNA that connects adjacent nucleosomes, bound by histone H1 to stabilize the structure.
• Chromatin Hierarchy: Nucleosomes ("beads-on-a-string", $10$ nm diameter) stack to form a $30$ nm chromatin fiber, which loops onto a non-histone protein scaffold ($300$ nm) to ultimately condense into metaphase chromosomes ($700$ nm).

• Nuclear Genome: Comprises the bulk of cellular DNA; contains non-coding introns interspersed between coding exons. Only about $2\%$ of the human genome ($3000$ Mb) codes for proteins, with the remainder consisting of repetitive sequences and regulatory elements.
• Organellar Genomes: Located in mitochondria and chloroplasts. These genomes are circular, double-stranded, lack introns, replicate semi-conservatively, and exhibit uniparental (maternal) inheritance.

In 1958, Matthew Meselson and Franklin Stahl proved semi-conservative replication in E. coli using nitrogen isotopes:

1. Bacteria were grown in a heavy nitrogen isotope ($^{15}\text{N}$) medium for several generations until all DNA was fully labeled.
2. The culture was transferred to a light nitrogen isotope ($^{14}\text{N}$) medium.
3. DNA was extracted at specific intervals and analyzed using CsCl density gradient centrifugation.
4. Results: Generation 0 yielded a single heavy band ($^{15}\text{N}$-$^{15}\text{N}$). Generation 1 (after 20 minutes) yielded a single hybrid band ($^{15}\text{N}$-$^{14}\text{N}$). Generation 2 (after 40 minutes) yielded two bands of equal intensity: one hybrid ($^{15}\text{N}$-$^{14}\text{N}$) and one light ($^{14}\text{N}$-$^{14}\text{N}$). This confirmed the semi-conservative model.

Replication requires a coordinated network of enzymes and proteins:

1. Initiation: Initiator proteins bind to the origin of replication (ori) and recruit helicase to unwind DNA, forming a replication bubble with two bidirectional replication forks.
2. Elongation: DNA polymerase III synthesizes DNA continuously on the leading strand ($5' \rightarrow 3'$ direction, moving toward the fork). On the lagging strand, synthesis is discontinuous, producing short Okazaki fragments ($1000-2000$ bp) away from the fork. Each fragment requires a new RNA primer.
3. Ligation: DNA Polymerase I replaces RNA primers with DNA, and DNA ligase seals the remaining nicks.
4. Termination: In circular prokaryotic chromosomes, replication forks meet at a specific terminus site opposite the origin, where termination proteins block helicase activity.

The flow of genetic information is unidirectional: $\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}$. In retroviruses, genetic information flows in reverse via reverse transcription (catalyzed by reverse transcriptase), converting viral RNA into complementary DNA (cDNA).

Transcription is catalyzed by a single DNA-dependent RNA Polymerase:

• Holoenzyme: Composed of the core enzyme ($\alpha_2\beta\beta'$) and the sigma ($\sigma$) factor, which is required for promoter recognition and initiation.
• Transcription Unit: Consists of a Promoter (containing the consensus sequence $\text{TATAAT}$ or Pribnow box at $-10$), an RNA-coding region, and a Terminator.
• Initiation: The holoenzyme binds the promoter, unwinds approximately $17$ bp of DNA to form a transcription bubble, and initiates RNA synthesis without a primer.
• Elongation: The core enzyme moves along the template strand, synthesizing RNA in the $5' \rightarrow 3'$ direction at a rate of $\sim 40$ nucleotides per second.
• Termination: Occurs via rho-dependent (requires Rho protein) or rho-independent (formation of a GC-rich hairpin loop followed by a poly-U tract) mechanisms.

Eukaryotic transcription is compartmentalized within the nucleus and utilizes three distinct RNA polymerases:

• RNA Polymerase I: Transcribes $28\text{S}$, $18\text{S}$, and $5.8\text{S}$ rRNAs.
• RNA Polymerase II: Transcribes pre-mRNA (heterogeneous nuclear RNA or hnRNA).
• RNA Polymerase III: Transcribes tRNA and $5\text{S}$ rRNA.

Eukaryotic pre-mRNA must undergo processing before nuclear export:

1. Capping: Addition of a $5'\text{-methylguanosine cap}$ ($5'\text{-methyl G cap}$) to protect the transcript from exonuclease degradation and facilitate ribosome binding.
2. Splicing: Removal of non-coding introns and ligation of coding exons, catalyzed by the spliceosome.
3. Polyadenylation: Addition of a $3'\text{-poly-A tail}$ ($80-250$ adenine residues) to stabilize the mRNA and assist in nuclear export.

George Gamow proposed that a triplet code ($4^3 = 64$ codons) is the minimum required to code for $20$ amino acids. Marshall Nirenberg, Heinrich Matthaei, and Har Gobind Khorana deciphered the code using synthetic homopolymers (e.g., poly-U translated to polyphenylalanine, establishing $\text{UUU} = \text{Phe}$).

1. Triplet Nature: Composed of three-nucleotide codons; $61$ codons specify amino acids, while $3$ (UAA, UAG, UGA) serve as stop signals.
2. Degeneracy: Most amino acids are specified by more than one codon (except methionine and tryptophan).
3. Unambiguous: Each codon specifies only one unique amino acid.
4. Non-overlapping & Commaless: Read continuously without punctuation or shared bases.
5. Universality: Nearly identical across all organisms, from bacteria to humans.
6. Dual Function of AUG: Acts as the start codon and codes for methionine.

This two-step activation process is catalyzed by aminoacyl-tRNA synthetase:

1. $\text{Amino Acid} + \text{ATP} \rightarrow \text{Aminoacyl-AMP} + \text{PP}_i$
2. $\text{Aminoacyl-AMP} + \text{tRNA} \rightarrow \text{Aminoacyl-tRNA} + \text{AMP}$
3. Wobble Hypothesis: Proposed by Francis Crick; allows the $5'$ base of the tRNA anticodon to form non-standard hydrogen bonds with the $3'$ base of the mRNA codon, reducing the minimum number of tRNAs required from $61$ to around $30-40$.

• Initiation: In prokaryotes, the $30\text{S}$ ribosomal subunit binds the Shine-Dalgarno sequence on mRNA. The initiator fMet-tRNAfmet binds to the start codon (AUG) at the ribosomal P-site. The $50\text{S}$ subunit then joins to form the active $70\text{S}$ initiation complex.
• Elongation: A charged aminoacyl-tRNA enters the vacant A-site. Peptidyl transferase catalyzes peptide bond formation between the amino acid at the A-site and the growing chain at the P-site. The ribosome then translocates three nucleotides in the $5' \rightarrow 3'$ direction, moving the uncharged tRNA to the E-site for exit.
• Termination: Occurs when a stop codon enters the A-site. Release factors recognize the stop codon, hydrolyze the ester bond linking the polypeptide to the tRNA, and trigger the dissociation of the ribosomal subunits.
• Polyribosomes: Multiple ribosomes translating a single mRNA molecule simultaneously to maximize protein synthesis efficiency.

• Point Mutations: Single base substitutions, classified as Transitions (purine to purine, e.g., $A \leftrightarrow G$, or pyrimidine to pyrimidine, e.g., $C \leftrightarrow T$) or Transversions (purine to pyrimidine or vice versa).
• Frameshift Mutations: Insertions or deletions of nucleotides not divisible by three, which alter the reading frame of all downstream codons.
• Spontaneous Mutations: Arise from replication errors or tautomeric shifts, where bases temporarily shift from their common keto/amino forms to rare enol/imino forms, causing mispairings (e.g., imino-guanine pairing with thymine).
• Induced Mutations: Caused by mutagens. Physical mutagens include ionizing radiation (X-rays) and non-ionizing radiation (UV rays, which induce thymine dimers). Chemical mutagens include alkylating agents (EMS) and base analogs (5-bromouracil).

Wild-type DNA replication requires precise keto-amino base pairing ($G \equiv C$ and $A = T$).

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Engineering Methodology & Mechanism: Spontaneous tautomeric shifts temporarily convert nitrogenous bases into rare enol or imino forms. For example, the imino form of guanine ($G^*$) mispairs with thymine ($T$) instead of cytosine ($C$). During subsequent replication cycles, this mispairing leads to a transition mutation where the original $G \equiv C$ pair is permanently replaced by an $A = T$ pair.

1. Base Excision Repair (BER): DNA glycosylase recognizes and removes a damaged base, creating an apurinic/apyrimidinic (AP site). An AP endonuclease cleaves the phosphodiester backbone, DNA polymerase fills the gap, and DNA ligase seals the nick.
2. Nucleotide Excision Repair (NER): Repairs bulky, helix-distorting lesions like UV-induced pyrimidine dimers. The UvrAB complex scans and detects the lesion, UvrC cleaves the damaged strand on both sides, helicase removes the oligonucleotide fragment, and DNA Polymerase I and ligase fill and seal the gap.
3. Mismatch Repair (MMR): Corrects replication errors. The MutS protein detects the mismatch, MutH binds to nearby hemimethylated GATC sites to distinguish the parental strand from the newly synthesized unmethylated strand, and an endonuclease excises the mismatch before repair synthesis occurs.

First described by Francois Jacob and Jacques Monod in 1961, an operon is a functional unit of genomic DNA containing a cluster of genes under the control of a single promoter and operator:

• Promoter: Binding site for RNA polymerase.
• Operator: DNA segment that acts as an on/off switch, bound by regulatory proteins.
• Regulator Gene: Codes for a repressor or activator protein.
• Structural Genes: Code for enzymes in a metabolic pathway.

The lac operon is an inducible system that regulates lactose metabolism:

• Structural Genes: lacZ (codes for $\beta$-galactosidase, which cleaves lactose into glucose and galactose), lacY (codes for $\beta$-galactoside permease, which facilitates lactose entry into the cell), and lacA (codes for $\beta$-galactoside transacetylase).
• Regulation in the Absence of Lactose: The regulatory gene lacI constitutively produces an active repressor protein that binds to the operator, physically blocking RNA polymerase and keeping transcription OFF.
• Regulation in the Presence of Lactose: Lactose is converted into allolactose (the inducer), which binds to the repressor. This induces a conformational change that inactivates the repressor, preventing it from binding to the operator. RNA polymerase is then free to transcribe the structural genes (ON state).
• Positive Control: Mediated by catabolite activator protein (CAP) bound to cAMP, which enhances RNA polymerase binding efficiency when glucose levels are low.