Control of Gene Expression in Prokaryotes

Making every protein at all times would be wasteful, but failing to make one when needed could be lethal. Bacteria solve this by wiring gene expression to environmental signals: produce lactose-digesting enzymes only when lactose is present, produce tryptophan-synthesizing enzymes only when tryptophan is scarce. The operon is the central mechanism, and it is one of the highest-yield topics on the MCAT.

Priority labels: Must know = cold; Know the logic = mechanism not names; Passage-level = recognize, don't memorize; Optional = skippable.

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The Operon Concept and the Jacob-Monod Model

What Is an Operon?

Must know

An operon is a cluster of functionally related genes transcribed together as a single polycistronic mRNA (one mRNA encoding multiple proteins). This is unique to prokaryotes — a clear contrast with eukaryotes, where genes are transcribed individually — and it means the genes are expressed all together or not at all.

The four components:

• Promoter — RNA polymerase binding site; where transcription starts.
• Operator — binding site for a regulatory protein (repressor or activator); sits at/near the promoter.
• Structural genes — encode the pathway's enzymes/proteins.
• Regulatory gene — encodes the repressor or activator. It is NOT part of the operon and is expressed constitutively (continuously, low level) to keep regulatory protein available.

The Jacob-Monod Model

Must know

In 1961, Jacob and Monod modeled the lac operon in E. coli (Nobel 1965): the first molecular explanation of gene regulation, showing that a small environmental molecule can control transcription by modulating protein binding to DNA.

Quick check: Why express metabolic genes as operons rather than individually?

Answer: All enzymes in a pathway are needed together and in proportional amounts, so one regulatory switch is efficient and ensures stoichiometric co-expression.

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The lac Operon: Negative Control by Repression

Structure of the lac Operon

Must know

The lac operon encodes three enzymes for importing and metabolizing lactose:

• lacZ → β-galactosidase: cleaves lactose into glucose + galactose; also converts a little lactose into allolactose, the actual inducer.
• lacY → lactose permease: imports lactose.
• lacA → transacetylase. Optional — lacA's exact role is rarely tested.

The regulatory gene lacI encodes the lac repressor, constitutively produced in small amounts.

How Negative Control Works

Must know

Negative control means a regulatory protein turns transcription off. When the lac repressor binds the operator, it blocks RNA polymerase → no transcription of lacZ, lacY, lacA.

Lactose absent: no allolactose → free repressor binds the operator tightly → transcription OFF. (Why make lactose enzymes with no lactose?)

Lactose present: β-galactosidase (trace amounts leak through) makes allolactose, the inducer. Allolactose binds the repressor, changing its shape so it falls off the operator → RNA polymerase transcribes the genes.

An operon that is normally OFF but turned ON when an inducer removes the repressor is an inducible operon.

$\text{No lactose} \Rightarrow \text{repressor binds operator} \Rightarrow \text{transcription OFF}$

$\text{Lactose present} \Rightarrow \text{allolactose binds repressor} \Rightarrow \text{repressor released} \Rightarrow \text{transcription ON}$

Constitutive Mutations and Merodiploids

Passage-level

Two mutations cause constitutive expression (always ON):

• lacI⁻ (trans-acting): non-functional repressor that can't bind the operator. Because it's a diffusible protein, a wild-type lacI⁺ copy on another DNA molecule can rescue it in trans.
• Oᶜ (operator constitutive, cis-acting): mutant operator that no repressor can bind. It affects only genes on the same DNA molecule, so a wild-type operator in trans does NOT rescue it.

Optional

A lacIˢ (super-repressor) binds the operator but not the inducer, so the operon stays OFF (uninducible) even with lactose — dominant in trans.

The cis/trans rule is tested with merodiploids (partial diploids, a second operon copy on an F′ plasmid, written as two genotypes split by a slash). The reliable method: go allele by allele — cis elements (promoter, operator) act only on their own DNA molecule; the lacI repressor is diffusible and acts on both operators.

Quick check: A mutant always makes β-galactosidase even without lactose; adding wild-type lacI on a plasmid corrects it. Is the mutation in lacI or the operator?

Answer: In lacI — a wild-type copy rescues it in trans. An operator (Oᶜ) mutant cannot be rescued in trans.

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Positive Control: Catabolite Activator Protein (CAP)

The Glucose Preference Problem

Must know

Removing the repressor is necessary but not sufficient for high lac transcription. E. coli prefers glucose, so a second layer of control enforces that preference. This layer is positive control — a protein that must actively assist RNA polymerase for high transcription.

Cyclic AMP and CAP

Must know

When glucose is low, cAMP is high; when glucose is high, cAMP is low.

CAP (Catabolite Activator Protein, also called CRP) binds cAMP. The CAP-cAMP complex binds upstream of the lac promoter and recruits/stabilizes RNA polymerase, boosting transcription initiation.

Know the logic

You don't need the adenylyl cyclase details — just glucose ↓ → cAMP ↑ → CAP active.

$\text{Low glucose} \Rightarrow \text{high cAMP} \Rightarrow \text{CAP-cAMP binds promoter region} \Rightarrow \text{transcription strongly activated}$

$\text{High glucose} \Rightarrow \text{low cAMP} \Rightarrow \text{CAP inactive} \Rightarrow \text{transcription low even if lactose is present}$

Integrating Both Signals

Must know

The lac operon has TWO inputs; evaluate both at once:

Glucose	Lactose	cAMP	Repressor	CAP	Transcription
Present	Absent	Low	Bound (blocking)	Inactive	~Zero
Present	Present	Low	Released	Inactive	Very low (basal)
Absent	Absent	High	Bound (blocking)	Active	~Zero
Absent	Present	High	Released	Active	High

Only the bottom row — lactose present AND glucose absent — runs the operon at full capacity. This is combinatorial control.

Glucose suppressing the genes for alternative sugars is called catabolite repression (the glucose effect).

Passage-level

The diauxic (biphasic) growth curve: in glucose + lactose, E. coli grows on glucose first (lac off), pauses (the diauxic shift/lag) as glucose runs out and cAMP rises to induce the lac operon, then grows on lactose. The common question — "what causes the lag?" — answer: the time to de-repress and induce the lac operon once glucose is depleted.

Quick check: Grown with both glucose and lactose present — is the lac operon highly expressed?

Answer: No. Allolactose releases the repressor, but glucose keeps cAMP low, so CAP is inactive and transcription stays low. High expression needs BOTH lactose present AND glucose absent.

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Gene Repression: The trp Operon

A Repressible Operon

Must know

The lac operon is inducible (normally off, turned on by substrate). The trp operon is the mirror image: a repressible operon, normally ON but turned off when the end product accumulates — keep making tryptophan until you have enough, then stop.

The trp operon encodes enzymes for tryptophan biosynthesis; the regulatory gene trpR encodes the trp repressor. Optional — the five enzyme genes (trpE, D, C, B, A) need not be memorized by name.

The key contrast with lac:

• lac repressor: active by default (binds operator); inactivated by its ligand (allolactose).
• trp repressor: inactive by default (an aporepressor); activated by its ligand. The corepressor is tryptophan itself — when Trp is high, it binds the aporepressor, which then binds the operator and blocks transcription.

$\text{Low [Trp]} \Rightarrow \text{aporepressor inactive} \Rightarrow \text{operator free} \Rightarrow \text{transcription ON}$

$\text{High [Trp]} \Rightarrow \text{Trp binds aporepressor} \Rightarrow \text{active repressor binds operator} \Rightarrow \text{transcription OFF}$

Both lac and trp use negative control (a repressor blocks transcription); the difference is that lac's ligand inactivates its repressor while trp's ligand activates its repressor.

Quick check: Intracellular tryptophan rises sharply. What happens to trp transcription, and why?

Answer: It turns OFF. Tryptophan is a corepressor: it binds the inactive aporepressor, making an active repressor that binds the operator — a repressible operon shutting down when its product is abundant.

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Comparing Inducible and Repressible Operons

Must know

Feature	lac (inducible)	trp (repressible)
Default state	OFF (repressor bound)	ON (repressor inactive)
Small molecule	Inducer (allolactose) inactivates repressor	Corepressor (tryptophan) activates repressor
Context	Catabolic (substrate → digest it)	Anabolic (product absent → synthesize it)
Control type	Negative (+ CAP positive)	Negative only

The inducer turns lac ON by removing its repressor; the corepressor turns trp OFF by activating its repressor. lac additionally uses positive control via CAP-cAMP.

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Common Confusions & Tricks

1. "Positive/negative control" = the regulatory protein's effect, not the outcome. Positive = an activator binds to increase transcription; negative = a repressor binds to decrease it. CAP is a positive regulator because it activates when bound.

2. Low glucose ≠ high lactose. Independent signals. Low glucose activates CAP; lactose removes the repressor. High-level expression needs both.

3. The lac inducer is allolactose, not lactose. Made by trace β-galactosidase. IPTG is a non-metabolizable synthetic inducer mimicking allolactose — recognize it in a passage.

4. trp vs. lac repressor logic — easy reversal error. lac repressor: active without ligand, INACTIVATED by allolactose (→ ON). trp repressor: inactive without ligand, ACTIVATED by tryptophan (→ OFF). Trick: Inducer and Inactivate both start with I — the inducer inactivates the repressor.

5. Cis vs. trans. The operator is just a binding site (cis): an Oᶜ mutation can't be rescued in trans. A lacI⁻ repressor is a diffusible protein (trans): a wild-type lacI on a plasmid can complement it.

6. CAP works by contacting RNA polymerase. It doesn't remove anything — it stabilizes polymerase at the promoter (unlike a repressor, which blocks access).

7. Two independent lac mechanisms (repressor + CAP). A mutant lacI with high glucose still gives low expression, because CAP is inactive — only one of two inputs is satisfied.

8. "Catabolite repression" is a misleading name. The actual mechanism is that absence of glucose activates transcription via CAP-cAMP.

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Key Takeaways

• An operon = promoter + operator + co-transcribed structural genes → polycistronic mRNA; prokaryote-specific.
• Jacob-Monod model: gene expression is regulated by small-molecule-controlled protein–DNA interactions.

lac operon (inducible; negative + positive control):

• lacZ, lacY, lacA → β-galactosidase, permease, transacetylase.
• lacI repressor (active by default) blocks the operator → OFF without lactose.
• Allolactose (not lactose) inactivates the repressor → ON with lactose.
• CAP-cAMP positive control: low glucose → high cAMP → CAP binds upstream → high transcription.
• Max expression needs BOTH lactose present AND glucose absent.
• Catabolite repression: glucose keeps cAMP/CAP inactive, suppressing alternative-sugar genes.

trp operon (repressible; negative control):

• Encodes Trp-biosynthesis enzymes; normally ON.
• Aporepressor inactive until tryptophan (corepressor) binds → active repressor blocks operator → OFF when Trp is abundant.

Inducible vs. repressible (both negative control):

• Inducible: normally OFF; ligand removes repressor → ON (lac; catabolic).
• Repressible: normally ON; ligand activates repressor → OFF (trp; anabolic).

Mutations (passage-level): lacI⁻ = non-functional repressor, constitutive, rescued in trans; Oᶜ = operator can't bind repressor, constitutive, NOT rescued in trans (cis-acting).