Operons, Mutation, and Mutagens – Comprehensive Study Notes

Inducible vs. Repressible Operons

• Core components common to all operons
– Promoter (binds RNA-polymerase)
– Operator (regulatory DNA sequence immediately downstream of promoter)
– Structural genes (encode the proteins of interest)
– Regulatory gene located elsewhere that produces a repressor protein

• Repressor protein options
– Bind operator → physically blocks RNA-polymerase → transcription OFF
– Bind a small metabolite (inducer or corepressor) → alters repressor shape → changes operator affinity

Lac (Lactose) Operon – Classic Inducible / Catabolic Model

• Native state = OFF
– Repressor synthesized in active form
– Binds operator; RNA-polymerase stalls at the protein “road-block.”

• Environmental lactose appears (the inducer)
– Repressor has higher affinity for lactose than for operator
– Lactose binding allosterically changes repressor shape → cannot bind DNA
– Operator is cleared → RNA-pol transcribes → mRNA translated into \approx 3 catabolic enzymes for lactose breakdown (exact names not required).

• Catabolism & shut-off
– Enzymes hydrolyze lactose until the last molecule is consumed
– Free repressor reverts to native conformation → re-binds operator → transcription halts (energy conservation).
– Makes sense for catabolic pathways: only synthesize enzymes when substrate present.

• Key descriptive phrases
– "OFF until ON"
– Inducible; almost always catabolic.

Trp (Tryptophan) Operon – Repressible / Anabolic Model

• Native state = ON (cell needs a trickle of Trp)
– Repressor synthesized inactive → cannot bind operator
– RNA-pol continuously transcribes the operon → mRNA encodes 5 biosynthetic enzymes (each consumes ATP).

• Excess tryptophan appears
– Trp acts as a corepressor
– Trp + repressor = active complex that now binds operator → transcription OFF.

• Consumption/resumption cycle
– Cell uses Trp for protein synthesis
– When intracellular [Trp] falls, it dissociates from repressor → repressor inactive again → transcription resumes
– “ON until OFF.”
– Repressible operons are typically anabolic: they build expensive molecules only as needed, in limited supply.

• Memory aid
– Inducer removes repressor; Corepressor enables repressor.

Gene Regulation vs. Evolutionary Change

• Short-term, minor or seasonal environmental shifts → alter gene expression (sweating, shivering)
– Regulatory changes are fast but not heritable.

• Long-term or permanent shifts → require genetic change
– Two universal mechanisms:
• Mutation (all life)
• Horizontal gene transfer (HGT) – only single-celled organisms (multicellular bodies cannot integrate DNA into every cell simultaneously).

Natural Selection Example 1 – Antibiotic Resistance in S. aureus

• Starting colony has mostly antibiotic-susceptible cells plus a few spontaneous resistant mutants.
• Add antibiotic → susceptible population killed → resistant mutants flourish.
• Remove antibiotic + time → susceptible cells out-compete because resistance costs energy → population reverts to susceptible majority.

Natural Selection Example 2 – Island Birds

• Original majority: thick-beaked birds suited for nuts/fruit.
• Pre-existing mutant with thin beak subsists but rare.
• Volcanic eruption destroys trees; food now small items in lava cracks.
• Thick-beak lineage starves; thin-beak mutants survive and become dominant.
• Key concept: environment selects among pre-existing mutants; it does not induce specific mutations.

Mutation Fundamentals

Origins

• Spontaneous (random replication errors, chemical instability) – vast majority.
• Induced by external mutagens (chemicals or radiation) – less common overall but can be potent.

Actual vs. Visible Mutation Rate (Richard Lenski’s insight)

• Actual rate: biochemical mis-incorporation frequency, roughly 1/10^{4} replications for most genes.
• Visible rate: proportion of mutants we observe; depends on gene essentiality.
– Mutation in essential gene (e.g., ribosomal protein) usually lethal → remains unseen.
– Mutation in non-essential or structural gene survives → visible frequency higher.
• Thus critical genes appear to mutate “less,” but lethality hides their events.

Point Mutations (Single-Base Substitutions)

Frameshift (Insertion/Deletion)

• Add or delete \pm 1 (or any non-multiple of 3) nucleotide → reading frame shifts.
• Every codon downstream changes → typically catastrophic: nonsense or severely misfolded protein.
• Analogy: “JAC KRA NUP THE …” after 1-letter deletion.

Chemical Mutagens

1. Base Modifiers (Most Potent)

• Chemically alter existing bases; mutation can arise without replication.
• Example: Alkylating agents – e.g., ethylene oxide (sterilization gas).
– Guanine → O-methyl-guanine mis-pairs with T
– Cellular repair (e.g., p53) replaces perceived mismatch, locking G→A transition into genome.
• Extremely effective sterilant but strong carcinogen; medical field aims to cut use by 95\%.

2. Base Analogs

• Structural mimics incorporated during replication.
• Example: 2-aminopurine (A analog) can pair with C → transition mutations.
• Weaker than modifiers because replication must coincide with presence.

3. Intercalating Agents

• Planar molecules slip between stacked bases → distort helix, cause strand breaks during unwinding.
• Lead to insertions/deletions (frameshifts).
• Example: Methylene blue (lab stain) – potent mutagen if ingested.
• Often lethal to cell before mutation becomes heritable, so paradoxically “less mutagenic” in practice.

Radiation Mutagens

Non-Ionizing – Ultraviolet (UV)

• UV-A, UV-B, UV-C (energy ↑, penetration ↓).
• Creates thymine dimers (covalent T-T bonds) → polymerase stalls; may cause frameshifts.

• Biological responses
– Humans: melanin + stratum corneum ≈ natural shields; Caucasians have \approx 70\% higher melanoma risk than darker-skinned groups.
– Bacteria: photolyase enzyme uses visible light to split dimers (light-dependent repair) → explains why UV disinfection is done in dark/closed systems.

• Clinical correlation
– UV-A deepest penetration → major driver of skin cancer.
– UV-B dominant in photo-aging (wrinkles).
– UV-C lowest penetration; primarily causes superficial burns; medical germicidal lamps use almost pure C to minimize carcinogenesis.

Ionizing – X, γ (Gamma), Cosmic Rays

• Energy sufficient to eject electrons → single- & double-strand breaks; “DNA spaghetti.”
• No known biological resistance (would require lead-like shielding).
• Applications
– X-rays: low-dose imaging; operators use lead/cement barriers.
– γ-rays: industrial & medical sterilization of pre-packaged items (replacing ethylene oxide). Requires thick concrete/lead chambers for containment.
– Cosmic rays: far higher energy; not practically harnessed.

Practical/Exam-Level Take-Home Points

• Operator + Repressor always present; difference lies in inducer (lac) vs corepressor (trp).
• Inducible operon = OFF → ON; usually catabolic.
• Repressible operon = ON → OFF; usually anabolic.

• Environment does not trigger new mutations; it selects pre-existing variants.

• Visible mutation rate inversely related to gene essentiality.

• Mutation taxonomy
– Substitutions: silent, missense, nonsense.
– Frameshift: insertion/deletion ≠ multiple of 3 bases.

• Mutagens
– Base modifiers > base analogs in potency.
– Intercalators specialize in frameshifts.
– UV → thymine dimers; repaired by photolyase + visible light.
– Ionizing radiation → DSBs, powerful sterilant but difficult to shield.

• Ethylene oxide vs γ-ray sterilization: know pros/cons, safety, penetration issues.

• Statistic/number recall
– Spontaneous mutation: \sim 1/10^{4} per gene per replication.
– Trp operon: 5 enzymes.
– Lac operon: 3 enzymes.
– Resistant S. aureus reverts when antibiotic removed + time.
– Caucasian melanoma risk \uparrow 70\% vs darker skin.

Use this outline as a complete substitute for the lecture transcript while preparing for the exam.