# Lecture 12: Regulation of Gene Expression: Prokaryotes

## Genetics

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Explain why gene regulation is essential for prokaryotic survival
2. Describe the operon model and its components (promoter, operator, structural genes, regulatory gene)
3. Analyze the lac operon as a model of inducible gene regulation with both negative and positive control
4. Analyze the trp operon as a model of repressible gene regulation
5. Explain the concept of attenuation in the trp operon
6. Describe global regulatory mechanisms including catabolite repression, regulons, and two-component signaling

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## Lecture Content

### I. Why Regulate Gene Expression?

Bacteria must rapidly adapt to constantly changing environmental conditions, including fluctuations in nutrient availability, exposure to stressors, and competition from other organisms. Constitutive expression of all genes would be energetically wasteful, so gene regulation allows bacteria to synthesize enzymes only when they are needed, conserve cellular resources, and respond rapidly to environmental signals. Most prokaryotic regulation occurs at the level of transcription initiation, which is the most efficient control point because it prevents unnecessary mRNA synthesis.

### II. The Operon Model

The operon model was proposed by Francois Jacob and Jacques Monod in 1961, work for which they received the Nobel Prize in 1965. An **operon** is a cluster of functionally related structural genes under coordinated control. Its components include the **structural genes**, which encode the enzymes or proteins of a metabolic pathway and are transcribed as a single polycistronic mRNA; the **promoter (P)**, where RNA polymerase binds and transcription initiates; the **operator (O)**, a DNA sequence where the repressor protein binds, typically overlapping the promoter; and the **regulatory gene**, which encodes the repressor protein and is often located nearby but is not part of the operon itself.

Regulation can be **negative** (a repressor protein binds the operator and blocks transcription) or **positive** (an activator protein enhances RNA polymerase binding and transcription). Operons can also be classified as **inducible** (normally off, turned on by an inducer, as in the lac operon) or **repressible** (normally on, turned off by a corepressor, as in the trp operon).

### III. The lac Operon: Inducible, Negative + Positive Control

The lac operon encodes the enzymes for lactose metabolism in *E. coli*: **lacZ** encodes beta-galactosidase, which cleaves lactose into glucose and galactose; **lacY** encodes permease, which transports lactose into the cell; and **lacA** encodes transacetylase, whose role in lactose metabolism is less clear. The regulatory gene **lacI** encodes the Lac repressor and is constitutively expressed from its own promoter.

**Negative control** is exerted by the Lac repressor. In the absence of lactose, the Lac repressor (a tetramer) binds the operator and blocks RNA polymerase, keeping transcription off. When lactose is present, allolactose (an isomer of lactose and the true inducer) binds the Lac repressor, causing a conformational change that prevents it from binding the operator, allowing transcription to proceed. IPTG (isopropyl-beta-D-thiogalactopyranoside) is a synthetic inducer commonly used in molecular biology because it is not cleaved by beta-galactosidase.

**Positive control** is provided by the CAP-cAMP system. Even with lactose present, the lac operon is not fully active unless glucose is absent, a phenomenon known as **catabolite repression (the glucose effect)**. When glucose is present, cAMP levels are low because adenylate cyclase is inhibited. When glucose is absent, cAMP levels rise, and **CAP (Catabolite Activator Protein, also called CRP)** binds cAMP to form a CAP-cAMP complex. This complex binds upstream of the promoter, enhancing RNA polymerase binding and driving high transcription. The lac operon requires both the inducer (allolactose) and the activator (CAP-cAMP) for maximal expression.

The four possible conditions can be summarized as follows. With glucose present and lactose absent, the repressor sits on the operator and cAMP is low, so the operon is off. With glucose and lactose both present, the repressor is released but cAMP remains low, yielding only basal expression. With glucose absent and lactose absent, CAP-cAMP binds upstream but the repressor blocks the operator, keeping the operon off. With glucose absent and lactose present, CAP-cAMP binds and the repressor is released, producing maximal expression.

<image>Panel A: Diagram of the lac operon showing the arrangement of lacI (with its own promoter), the main promoter, CAP binding site, operator, and structural genes lacZ, lacY, lacA. Four panels showing the operon state under each of the four glucose/lactose conditions, with repressor, CAP-cAMP, and RNA polymerase positions indicated. Panel B: Molecular detail of allolactose binding to the Lac repressor causing a conformational change that prevents DNA binding, showing the repressor as a tetramer with the inducer binding domains. Panel C: Graph showing beta-galactosidase expression levels under the four conditions (glucose+/lactose-, glucose+/lactose+, glucose-/lactose-, glucose-/lactose+) as a bar chart demonstrating the hierarchy of regulation.</image>

### IV. Lac Operon Mutant Analysis

Mutations in lac operon components reveal the underlying logic of gene regulation. A **lacI-** (loss-of-function repressor) mutation causes constitutive expression regardless of lactose, keeping the operon always on. A **lacIs** (super-repressor) mutation produces a repressor that cannot bind allolactose and therefore remains permanently bound to the operator, keeping the operon always off; this mutation is dominant in a diploid. A **lacOc** (operator constitutive) mutation produces an operator that cannot bind the repressor, causing constitutive expression. Because the operator affects only the operon on the same DNA molecule, this is a cis-acting mutation. A **lacP-** (promoter mutation) prevents RNA polymerase from binding, keeping the operon always off, and is also cis-acting.

The distinction between **cis** and **trans** effects, demonstrated with partial diploids (merodiploids), is fundamental to understanding gene regulation. Operator and promoter mutations are cis-acting because they affect only the adjacent genes on the same DNA molecule. The repressor gene (lacI) is trans-acting because the protein it encodes can diffuse through the cell and act on any copy of the operator. The lacIs super-repressor is trans-dominant over lacI+ because it binds the operator even in the presence of inducer.

### V. The trp Operon: Repressible, Negative Control

The trp operon encodes the five enzymes required for tryptophan biosynthesis (trpE, trpD, trpC, trpB, and trpA). As a repressible operon, it is normally on because the biosynthetic enzymes are needed unless tryptophan is supplied externally. The trpR gene encodes the aporepressor in its inactive form. When tryptophan is abundant, tryptophan acts as a **corepressor** by binding the aporepressor to form the active holorepressor, which then binds the operator and shuts off transcription. When tryptophan is scarce, the aporepressor alone cannot bind the operator and transcription proceeds. This logic is the opposite of the lac operon: in the trp operon, the small molecule (tryptophan) turns the operon off rather than on.

### VI. Attenuation in the trp Operon

Attenuation provides a second level of regulation that fine-tunes trp operon expression. This mechanism operates in the 5' leader region of the trp mRNA, upstream of the structural genes. The leader sequence contains a short open reading frame encoding a 14-amino-acid **leader peptide** that includes two tandem tryptophan codons. The leader RNA can form alternative stem-loop structures: regions 3-4 form a transcription terminator hairpin followed by a poly-U stretch, while regions 2-3 form an anti-terminator structure.

The mechanism depends on the coupling of transcription and translation in prokaryotes. When tryptophan is abundant, charged Trp-tRNA is plentiful, so the ribosome translates the leader peptide efficiently and covers region 2, allowing regions 3-4 to form the terminator hairpin. Transcription terminates prematurely (attenuation). When tryptophan is scarce, the ribosome stalls at the tandem Trp codons due to insufficient Trp-tRNA and covers region 1, allowing regions 2-3 to form the anti-terminator. Since the 3-4 terminator cannot form, transcription continues into the structural genes. Attenuation alone can reduce trp operon expression approximately 8-10 fold, and combined with repression, total regulation reaches 600-700 fold. Attenuation is not possible in eukaryotes because transcription and translation are spatially and temporally separated by the nuclear envelope.

<image>Panel A: Diagram of trp operon attenuation mechanism showing the leader region with regions 1, 2, 3, 4 labeled, the two Trp codons in the leader peptide, and two scenarios: (left) high Trp — ribosome translates quickly, covers region 2, regions 3-4 form terminator hairpin, transcription stops; (right) low Trp — ribosome stalls at Trp codons in region 1, regions 2-3 form anti-terminator, transcription continues. Panel B: Comparison table of the lac operon and trp operon showing their regulatory logic: inducible vs. repressible, negative and positive control elements, effector molecules, and conditions for ON and OFF states. Panel C: Diagram of the trp operon showing the regulatory gene trpR, promoter, operator, leader/attenuator region, and structural genes trpE-trpA, with tryptophan binding to the aporepressor to form the holorepressor that binds the operator.</image>

### VII. Global Regulatory Mechanisms

Beyond individual operons, bacteria employ several global regulatory strategies. **Regulons** are groups of operons or genes controlled by the same regulatory protein. The CAP-cAMP regulon, for example, activates many operons for alternative carbon source utilization when glucose is absent. The heat shock regulon uses sigma-32 (RpoH) to direct RNA polymerase to heat shock gene promoters when the cell encounters thermal stress.

**Alternative sigma factors** allow bacteria to redirect RNA polymerase to entirely different sets of promoters. Sigma-70 (RpoD) handles housekeeping genes, sigma-32 (RpoH) directs heat shock responses, sigma-54 (RpoN) controls nitrogen metabolism, sigma-S (RpoS) governs the stationary phase and stress response, and sigma-28 (FliA) regulates flagellar genes.

**Two-component regulatory systems** are a widespread mechanism for environmental sensing in bacteria. A **sensor kinase**, a membrane protein, detects an environmental signal and autophosphorylates on a histidine residue. The phosphate group is then transferred to an aspartate residue on a **response regulator** in the cytoplasm, activating it to bind DNA and regulate transcription. The EnvZ-OmpR system, which senses osmolarity and regulates porin gene expression, is a classic example. **Quorum sensing** allows bacteria to detect population density through signaling molecules called autoinducers. When cell density is high enough that autoinducer concentration reaches a threshold, gene expression is activated, coordinating group behaviors such as biofilm formation, virulence factor production, and bioluminescence.

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