Gene Control – Cambridge A‑Level Biology (9700)
1. Mapping to the Cambridge A‑Level Syllabus
| Syllabus requirement (Topic) | Notes section that satisfies it | Action needed (if any) |
|---|
| 16 – Inheritance: Mendelian genetics, chromosome behaviour, linkage, sex‑linked traits | Link added in “Link to syllabus” (see below) – brief sub‑section on classical genetics | Insert concise overview of monohybrid/dihybrid crosses and linkage maps |
| 17 – Selection & Evolution: role of gene regulation in adaptation | Section 4 (Prokaryotic) and Section 5 (Eukaryotic) discuss regulatory mutations and evolutionary advantage | Highlight examples such as lactose utilisation in gut bacteria and colour‑pattern changes in insects |
| 19 – Genetic Technology: DNA fingerprinting, PCR, recombinant DNA, GM crops, CRISPR, RNA‑i | Sections 6 (Post‑transcriptional), 7 (Epigenetics), 8 (Biotechnological applications) | Add short paragraph on PCR and DNA fingerprinting under “Genetic technology” |
2. Structural Genes vs. Regulatory Genes
| Feature | Structural genes | Regulatory genes |
|---|
| General definition |
|---|
| Primary product | Functional protein – enzyme, transporter, structural component, etc. | Regulatory molecule – repressor, activator, transcription factor, or regulatory RNA. |
| Typical location in a prokaryotic operon | Down‑stream of the promoter; co‑transcribed as part of a poly‑cistronic mRNA. | Often separate (e.g. lacI) or embedded in the promoter/operator region. |
| Role in the cell | Directly participates in metabolic pathways or structural assemblies. | Controls when, where and how much the structural genes are expressed. |
| Regulation of its own expression | May be constitutive or subject to control by regulatory proteins. | Can be constitutive (e.g. housekeeping repressor) or itself regulated by feedback loops. |
3. Repressible Enzymes vs. Inducible Enzymes
| Aspect | Repressible enzyme (usually anabolic) | Inducible enzyme (usually catabolic) |
|---|
| Default transcriptional state | ON – enzyme is synthesised unless repression occurs. | OFF – enzyme is not synthesised until an inducer is present. |
| Regulating signal | End‑product (co‑repressor) binds to a repressor → repressor–DNA complex → transcription off. | Substrate (inducer) binds to a repressor (or activates an activator) → repressor released → transcription on. |
| Classic example | Threonine synthase in the threonine biosynthetic pathway (repressed by threonine). | β‑galactosidase in the lac operon (induced by allolactose or IPTG). |
| Feedback type | Negative feedback (product inhibition). | Positive regulation (induction). |
| Quantitative illustration (Cambridge‑style) | Enzyme activity falls hyperbolically with product concentration; at 5 mM threonine activity ≈ 20 % of maximum. | β‑galactosidase activity rises steeply with inducer; at 1 mM IPTG activity ≈ 90 % of the maximum observed at 5 mM. |
4. Prokaryotic Gene Regulation (Operons)
- Operon structure: promoter – operator – structural genes (e.g. lacZYA).
- Repressor‑binding model:
- In the absence of inducer, the repressor protein binds the operator → RNA polymerase blocked → genes OFF.
- Inducer (allolactose or IPTG) binds the repressor, causing a conformational change → repressor releases → transcription ON.
- Repressible operon example – the trp operon:
- When tryptophan levels are high, tryptophan acts as a co‑repressor, binds the Trp repressor, and the complex binds the operator → synthesis of tryptophan‑biosynthetic enzymes OFF.
- Catabolite repression – cAMP‑CRP complex required for high expression of the lac operon; low glucose → high cAMP → activation; high glucose → low cAMP → reduced expression even with inducer present.
- Experimental relevance – inducible promoters (T7‑lac, araBAD) are routinely used for recombinant protein production.
5. Eukaryotic Gene Regulation
- Promoter architecture
- Core promoter (TATA box, Initiator) – binding site for RNA polymerase II.
- Proximal promoter elements – binding sites for specific transcription factors (TFs).
- Enhancers & silencers
- Can lie thousands of base pairs upstream or downstream.
- DNA looping brings bound activator or repressor proteins into contact with the basal transcription machinery.
- Transcription factors
- General TFs (TFIID, TFIIH, etc.) – required for basal transcription.
- Specific TFs – respond to hormones, growth factors, developmental cues (e.g. steroid‑receptor complexes, p53).
- Chromatin remodelling
- Histone acetyltransferases (HATs) add acetyl groups → open chromatin → active transcription.
- Histone deacetylases (HDACs) remove acetyl groups → condensed chromatin → repression.
- Co‑activators & co‑repressors
- Co‑activators bridge TFs to the basal machinery (e.g. Mediator complex).
- Co‑repressors recruit HDACs or DNA‑methyltransferases.
6. Post‑Transcriptional & RNA‑Based Regulation
- RNA processing – 5′ capping, splicing (removal of introns), 3′ poly‑A tail; alternative splicing generates protein isoforms.
- mRNA stability – AU‑rich elements in the 3′ UTR target transcripts for rapid degradation; stabilising proteins (e.g. HuR) bind and protect them.
- microRNA (miRNA) & small interfering RNA (siRNA)
- Short (~21 nt) RNAs incorporated into RISC bind complementary mRNA.
- Perfect complementarity → cleavage (siRNA); partial complementarity → translational repression (miRNA).
- Riboswitches (mainly in bacteria, some eukaryotic organelles) – metabolite‑binding RNA elements that alter transcription termination or translation initiation.
- PCR & DNA fingerprinting (Topic 19) – brief note: PCR amplifies a specific DNA fragment; STR analysis provides a genetic “fingerprint” used in forensic science and paternity testing.
7. Epigenetic Control
| Mechanism | Effect on gene expression | Cambridge‑style example |
|---|
| DNA methylation (addition of CH₃ to cytosine in CpG islands) | Usually silences transcription by preventing TF binding or recruiting methyl‑binding proteins. | X‑chromosome inactivation in mammals. |
| Histone modification (acetylation, methylation, phosphorylation) | Acetylation → open chromatin → active; methylation can activate (H3K4me) or repress (H3K9me) depending on residue. | H3K9 methylation associated with heterochromatin in pericentromeric regions. |
| Chromatin‑remodelling complexes (SWI/SNF, NuRD) | Move or evict nucleosomes to expose or hide promoter DNA. | Regulation of the β‑globin gene cluster during embryonic → adult switching. |
8. Applications in Modern Biotechnology
- Inducible expression systems – T7‑lac promoter in E. coli; IPTG induces high‑level recombinant protein production.
- CRISPR‑Cas genome editing – guide RNA directs Cas nuclease to a specific locus; can knock‑out a regulatory gene, insert a promoter, or correct a disease‑causing mutation.
- RNA‑i therapeutics – synthetic siRNA used to silence viral or oncogenic transcripts (e.g. patisiran for transthyretin amyloidosis).
- Gene‑therapy vectors – viral (AAV) or plasmid vectors carry a functional structural gene under the control of a tissue‑specific regulatory element.
- Epigenetic drugs – HDAC inhibitors (vorinostat, romidepsin) reactivate silenced tumour‑suppressor genes in certain cancers.
- DNA fingerprinting & PCR – forensic identification, paternity testing, detection of genetically modified organisms (GMOs) in food.
9. Key Points to Remember
- Structural genes code for the functional product; regulatory genes code for molecules that control the expression of structural genes.
- Repressible enzymes are switched OFF by the end‑product (negative feedback); inducible enzymes are switched ON by a substrate or analogue (positive regulation).
- Prokaryotic operons (e.g. lac, trp) illustrate how a single regulator can coordinate several structural genes.
- Eukaryotic transcription requires a core promoter, specific transcription factors, and often distant enhancers or silencers; chromatin state is a decisive third layer.
- Post‑transcriptional mechanisms (splicing, mRNA stability, miRNA/siRNA) and epigenetic modifications add further levels of control.
- Understanding these control systems underpins many biotechnological tools such as inducible expression vectors, CRISPR editing, RNA‑i therapeutics, and epigenetic drugs.
10. Sample AO3 Experimental Question
Design an experiment to investigate how the concentration of an inducer affects the activity of an inducible enzyme in Escherichia coli. Include the following points.
- Choice of system – lac operon; β‑galactosidase activity measured by ONPG (o‑nitrophenyl‑β‑D‑galactopyranoside) hydrolysis.
- Culture preparation – inoculate identical overnight cultures, dilute to the same OD₆₀₀ (≈0.1), grow to mid‑log phase (OD₆₀₀≈0.5) at 37 °C with shaking.
- Inducer gradient – add IPTG to final concentrations of 0, 0.1, 0.5, 1, and 5 mM; incubate for a fixed induction period (e.g. 15 min).
- Stopping the reaction – add 1 M Na₂CO₃ to each tube to raise pH and stop β‑galactosidase activity.
- Assay – measure absorbance of the yellow product (o‑nitrophenol) at 420 nm using a spectrophotometer.
- Calculations – determine specific activity (µmol ONP min⁻¹ mg⁻¹ protein) using the Miller formula; plot specific activity versus IPTG concentration.
- Interpretation
- Identify the concentration at which the curve plateaus (promoter saturation).
- Discuss possible catabolite repression if glucose is present in the medium.
- Explain how the shape of the curve demonstrates positive regulation (induction).
11. Suggested Diagrams for Revision
- Prokaryotic operon schematic – promoter, operator, structural genes; show repressor bound vs. inducer‑bound state.
- Eukaryotic transcription initiation – TATA box, general transcription factors, a bound enhancer‑activator complex, and an acetylated nucleosome.
- miRNA‑mediated silencing – miRNA‑RISC complex bound to the 3′ UTR of a target mRNA, preventing translation.
- DNA methylation – methyl groups on CpG islands blocking transcription‑factor binding.
- CRISPR‑Cas9 editing – guide RNA, Cas9 nuclease, double‑strand break, and repair by homology‑directed repair (HDR) or non‑homologous end joining (NHEJ).