explain genetic control of protein production in a prokaryote using the lac operon (knowledge of the role of cAMP is not expected)

Gene Control in Prokaryotes – The lac Operon

Why it matters for Cambridge AS/A‑Level (9700)

• Demonstrates transcriptional regulation, a core sub‑topic of 6 Nucleic acids & protein synthesis.
• Provides a model for inducible promoters used in 19 Genetic technology (e.g. IPTG‑induced expression vectors).
• Shows how bacteria conserve energy by expressing genes only when required – linked to the syllabus themes “Organisms in their environment” and “Observation and experiment”.
• Forms the basis of a practical (β‑galactosidase assay) that develops AO3 experimental skills.

1. The lac Operon – an overview

The lac operon of Escherichia coli is an inducible cluster of genes that enables the bacterium to use lactose as a carbon source only when lactose is present. It is a classic example of a single regulatory system that can switch the synthesis of several proteins on or off at the transcriptional level.

2. Components and their functions

3. Flow of information – DNA → RNA → protein

1. DNA (lac operon) – the genetic template.
2. Transcription – RNA polymerase binds the promoter and synthesises a single polycistronic mRNA that contains the coding sequences for lacZ, lacY and lacA (provided the operator is free).
3. Translation – ribosomes initiate at the start codon of each gene on the polycistronic mRNA, producing three separate enzymes: β‑galactosidase, permease and transacetylase.
4. Function – the enzymes allow uptake and breakdown of lactose, providing carbon and energy for the cell.

This illustrates the central dogma and shows how transcriptional control can regulate an entire metabolic pathway.

4. Regulation of the lac operon

4.1 Operon OFF – no lactose

• LacI repressor is produced continuously from the lacI gene.
• LacI binds tightly to the operator, physically blocking RNA polymerase from accessing the promoter.
• Result: no transcription of lacZ, lacY or lacA → no enzymes are made.

4.2 Operon ON – lactose present

1. Lactose diffuses into the cell at low levels.
2. β‑galactosidase catalyses the isomerisation of a small amount of lactose to allolactose (the true inducer):

   
   lactose ⇌ allolactose

3. Allolactose binds to LacI, inducing a conformational change that reduces its affinity for the operator.
4. The operator becomes unoccupied; RNA polymerase can now bind the promoter and transcribe the three structural genes.
5. β‑galactosidase production rises, hydrolysing more lactose → more allolactose is generated – a positive feedback loop.

4.3 Catabolite repression (glucose effect) – optional syllabus point

• When glucose is abundant, intracellular cAMP levels are low; the cAMP‑CAP complex does not form.
• Without the cAMP‑CAP activator bound to the CAP site, transcription from the lac promoter is weak even if allolactose is present.
• Thus, the operon is maximally expressed only when both lactose (inducer) is present and glucose (preferred carbon source) is scarce.
• Cambridge AS/A‑Level does not require detailed biochemistry of cAMP, but the concept of catabolite repression should be mentioned as an additional layer of control.

4.4 Comparison with eukaryotic regulation (brief)

• Prokaryotic operons are polycistronic and regulated mainly at the transcriptional level (repressor/activator binding).
  

• Eukaryotic genes are usually monocistronic; regulation often involves chromatin modification, enhancers, and post‑transcriptional control (splicing, RNA stability).

5. Linking the lac operon to the Cambridge syllabus

6. Practical application – AO3 skill development

Experiment: Measuring β‑galactosidase activity with ONPG (ortho‑nitrophenyl‑β‑D‑galactopyranoside)

6.1 Objective

Compare enzyme activity in E. coli cultures grown (a) without inducer, (b) with lactose, and (c) with the non‑metabolizable inducer IPTG.

6.2 Materials

• E. coli strain (lac⁺)
• LB broth, sterile flasks
• Lactose (1 % w/v) and IPTG (0.5 mM)
• ONPG solution (4 mg ml⁻¹)
• Sodium carbonate (stop solution)
• Spectrophotometer (420 nm), cuvettes
• Ice bath, water bath (37 °C), centrifuge
• Protein assay kit (e.g., Bradford) for normalisation

6.3 Method (outline)

1. Inoculate three 50 ml flasks with identical inocula of overnight culture.
2. Add (a) no inducer, (b) 1 % lactose, (c) 0.5 mM IPTG. Grow at 37 °C, 200 rpm until mid‑log phase (OD₆₀₀ ≈ 0.5).
3. Harvest 1 ml of each culture, keep on ice, and centrifuge (5 min, 4 °C).
4. Resuspend pellets in 0.1 ml Z buffer, lyse by adding 0.05 ml 0.1 % SDS and 0.05 ml chloroform; vortex briefly.
5. Add 0.5 ml ONPG, start a timer, and incubate at 37 °C. Record the time taken for the yellow colour to appear.
6. Stop the reaction with 0.5 ml 1 M Na₂CO₃, mix, and measure absorbance at 420 nm.
7. Determine protein concentration of each lysate (Bradford) and calculate specific activity:

   
   β‑gal units = (ΔA₄₂₀ × 1000) / (time min × protein mg)

8. Plot specific activity (units mg⁻¹) against inducer type (none, lactose, IPTG).

6.4 Controls and evaluation

• Negative control: heat‑killed cells (boiled 5 min) to confirm that colour development is enzyme‑dependent.
• Positive control: a strain known to over‑express lacZ (e.g., lacI⁻ mutant).
• Standardise cell density (OD₆₀₀) before lysis to minimise variation.
• Potential sources of error: temperature fluctuations, incomplete lysis, inaccurate protein quantification, and timing of colour development.
• Statistical analysis – calculate mean, standard deviation (n ≥ 3) and use a t‑test to assess significance of differences between treatments.
• Ethical note – work with non‑pathogenic E. coli; follow biosafety level 1 guidelines.

7. Biotechnological relevance – the lac promoter in recombinant DNA work

• lac promoter (P_lac) is an inducible promoter widely used in cloning vectors.
• A typical expression vector contains:

  • origin of replication (high copy number for large protein yields),
  • selectable marker (e.g., ampicillin resistance),
  • multiple cloning site downstream of P_lac,
  • the lacI gene (often on a compatible helper plasmid) to supply repressor.

• During growth, LacI binds the operator and transcription is repressed. Adding IPTG releases LacI, allowing high‑level transcription of the gene of interest.
• Advantages: tight regulation, rapid induction, no need for lactose metabolism, scalable for industrial protein production.
• Limitations: leaky expression in some strains, possible toxicity of the recombinant protein before induction, requirement for an additional plasmid or chromosomal lacI source.

8. Summary – key points for exam

• Identify the three structural genes (lacZ, lacY, lacA) and the enzymes they encode.
• Explain the roles of the promoter (RNA polymerase binding) and operator (repressor binding).
• Describe how LacI blocks transcription in the absence of inducer.
• State why allolactose, not lactose itself, is the natural inducer and give the simple conversion reaction.
• Outline the sequence of events that leads to enzyme synthesis when lactose is present, including the positive feedback loop.
• Recall catabolite repression: high glucose → low cAMP → weak transcription even with allolactose.
• Summarise the practical (ONPG assay) – purpose, key steps, controls, and data analysis.
• Describe the use of the lac promoter in recombinant DNA vectors, noting advantages and limitations.