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Lecture 23: Regulation of Gene Expression in Prokaryotes

General Biology I — Molecular & Cellular

Learning Objectives

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

1. Explain why gene regulation is essential for prokaryotic survival and metabolic efficiency
2. Describe the operon model and identify its structural and regulatory components
3. Compare and contrast the lac operon (inducible) and trp operon (repressible) as model regulatory systems
4. Explain the role of positive and negative regulation, including catabolite repression and the CAP-cAMP system
5. Describe additional mechanisms of prokaryotic gene regulation, including attenuation and riboswitches

Lecture Content

I. Why Regulate Gene Expression?

Prokaryotes inhabit rapidly changing environments where nutrient availability, temperature, osmolarity, and exposure to toxins can shift within minutes. Synthesizing every possible protein at all times would be enormously wasteful--each unnecessary mRNA and protein costs the cell ATP and biosynthetic resources that could be directed elsewhere. Gene regulation solves this problem by enabling cells to produce proteins only when they are needed, matching the cell's molecular output to its current circumstances.

Not all genes are regulated equally. Constitutive genes (housekeeping genes), such as those encoding ribosomal RNA or the enzymes of glycolysis, are expressed at a relatively constant level because their products are always required. Regulated genes, by contrast, are turned on or off--or modulated between these extremes--in response to specific environmental signals. Regulation can theoretically occur at any step in gene expression, but in prokaryotes transcriptional regulation predominates. This makes energetic sense: by preventing transcription of an unneeded gene, the cell avoids the cost of synthesizing both the mRNA and the protein. Post-transcriptional, translational, and post-translational regulation also occur in prokaryotes but are less prevalent.

II. The Operon Model

The operon model, proposed by Francois Jacob and Jacques Monod in 1961 (for which they shared the Nobel Prize in 1965), describes how prokaryotes coordinate the expression of functionally related genes. An operon is a cluster of genes that are transcribed together as a single polycistronic mRNA from a shared promoter. This arrangement is unique to prokaryotes; eukaryotic mRNAs are monocistronic, each encoding only one protein.

An operon consists of several components. The structural genes encode the enzymes or proteins for a particular metabolic pathway and are arranged in tandem along the DNA. The promoter (P) is the DNA sequence where RNA polymerase binds to initiate transcription. The operator (O), located between or overlapping the promoter and the structural genes, is the binding site for the repressor protein. The regulatory gene, often located upstream of the operon and transcribed independently from its own promoter, encodes the repressor protein that controls operon expression. Because the mRNA produced from an operon is polycistronic, it contains multiple open reading frames, each with its own Shine-Dalgarno sequence for independent ribosome binding and translation.

<image>A diagram of the general operon structure in prokaryotes. The top shows a linear stretch of DNA with the regulatory gene (with its own promoter) on the left, followed by the operon promoter (P), the operator (O), and three structural genes (Gene A, Gene B, Gene C) in tandem. Below, two states are shown. State 1 (Repressed): The repressor protein (encoded by the regulatory gene) binds to the operator, physically blocking RNA polymerase from transcribing the structural genes. State 2 (Active): The operator is unoccupied, and RNA polymerase transcribes all three structural genes into a single polycistronic mRNA, which is then translated into three separate proteins at independent ribosome binding sites.</image>

III. The lac Operon -- An Inducible System

The lac operon controls the metabolism of lactose in E. coli and serves as the paradigmatic example of an inducible gene regulatory system. It contains three structural genes: lacZ, encoding beta-galactosidase (which cleaves lactose into glucose and galactose); lacY, encoding permease (which transports lactose into the cell); and lacA, encoding transacetylase (whose function is less central to lactose metabolism). The regulatory elements include lacI, the regulatory gene encoding the lac repressor (a tetrameric protein), and the operon's promoter (P_lac) and operator (O_lac).

Negative Regulation of the lac Operon

In the absence of lactose, the lac repressor binds tightly to the operator. This physical obstruction prevents RNA polymerase from transcribing past the operator, effectively shutting down expression of the structural genes. Only basal, very low levels of the lac enzymes are produced.

When lactose is present, the small amount that enters the cell through residual permease is converted by beta-galactosidase into allolactose, an isomer that serves as the true inducer. Allolactose binds to the lac repressor and induces a conformational change (an example of allosteric regulation) that causes the repressor to release from the operator. With the operator clear, RNA polymerase can now transcribe the structural genes, and enzyme levels increase approximately 1000-fold. This is the defining behavior of an inducible operon: the substrate itself triggers expression of the genes needed for its metabolism. In laboratory settings, the synthetic inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) is often used because it binds and inactivates the repressor but, unlike allolactose, is not hydrolyzed by beta-galactosidase, providing stable, persistent induction.

Positive Regulation -- Catabolite Repression and CAP-cAMP

Even when lactose is available, E. coli prefers glucose as its carbon source. The mechanism that enforces this preference is called catabolite repression, and it operates through the catabolite activator protein (CAP), also known as CRP (cAMP receptor protein), in conjunction with the second messenger cAMP.

When glucose levels are high, the enzyme adenylate cyclase is inhibited, so cAMP levels remain low. Without cAMP, CAP cannot bind to DNA, and RNA polymerase binds only weakly to the lac promoter. Even if lactose is present and the repressor has been removed, transcription remains at a low, basal level. When glucose levels are low, adenylate cyclase is active and cAMP accumulates. The CAP-cAMP complex forms and binds to a specific site upstream of the lac promoter, bending the DNA and dramatically enhancing RNA polymerase recruitment. Transcription is now strongly activated.

The lac operon therefore has four possible regulatory states:

Maximal expression requires both the absence of glucose (so that CAP-cAMP activates transcription) and the presence of lactose (so that allolactose removes the repressor). This dual-input logic ensures that the cell invests in lactose metabolism only when glucose is unavailable and lactose is at hand.

<image>A four-panel diagram showing the four regulatory states of the lac operon. Panel 1 (Glucose present, No lactose): The repressor is bound to the operator, and CAP-cAMP is absent from the CAP binding site — operon is OFF. Panel 2 (Glucose present, Lactose present): Allolactose removes the repressor from the operator, but CAP-cAMP is absent because cAMP levels are low — only low-level transcription occurs. Panel 3 (No glucose, No lactose): CAP-cAMP is bound upstream of the promoter, but the repressor is on the operator — operon is OFF. Panel 4 (No glucose, Lactose present): CAP-cAMP is bound upstream and the repressor is removed by allolactose — RNA polymerase is strongly recruited, and maximal transcription of lacZ, lacY, and lacA occurs.</image>

IV. The trp Operon -- A Repressible System

The trp operon controls the biosynthesis of tryptophan in E. coli and illustrates the opposite regulatory logic from the lac operon. It contains five structural genes (trpE, trpD, trpC, trpB, trpA) encoding the enzymes that synthesize tryptophan from the precursor chorismate. Because the cell needs to manufacture tryptophan whenever it is not available from the environment, the trp operon is normally ON.

Negative Regulation

The trp repressor, encoded by the trpR gene, is synthesized in an inactive form called the aporepressor. On its own, the aporepressor cannot bind the operator. When tryptophan is abundant, however, tryptophan molecules act as corepressors--they bind to the aporepressor and induce a conformational change that enables it to recognize and bind the operator, blocking transcription. When tryptophan is scarce, no corepressor is available, the repressor remains inactive, and RNA polymerase freely transcribes the structural genes. This is a repressible operon: the end product of the biosynthetic pathway shuts off its own synthesis, representing feedback regulation at the level of gene expression.

Attenuation

The trp operon employs a second, finer-grained regulatory mechanism called attenuation that adjusts expression according to tryptophan levels with greater precision than repression alone. This mechanism operates within the leader sequence--a region of the 5' UTR upstream of the first structural gene. The leader contains a short open reading frame encoding a leader peptide that includes two tandem tryptophan codons. Critically, the leader mRNA can fold into alternative stem-loop (hairpin) structures with different functional consequences.

When tryptophan levels are high and charged Trp-tRNAs are abundant, the ribosome translates the leader peptide without pausing. As the ribosome moves through region 2 of the leader, it prevents region 2 from pairing with region 3. Regions 3 and 4 are therefore free to base-pair, forming a terminator hairpin followed by a run of uracils. This structure causes RNA polymerase to dissociate, and transcription terminates before reaching the structural genes.

When tryptophan levels are low and Trp-tRNAs are scarce, the ribosome stalls at the tandem tryptophan codons in region 1. With the ribosome stalled, region 2 is free to pair with region 3, forming an antiterminator hairpin. Because region 3 is now sequestered, the terminator hairpin (which requires region 3 paired with region 4) cannot form. RNA polymerase reads through the leader and continues transcribing the full operon. Attenuation thus provides a graded, proportional response: the lower the tryptophan supply, the more transcripts reach the structural genes.

<image>A two-panel diagram of the attenuation mechanism in the trp operon. Panel A (High tryptophan): The ribosome translates the leader peptide rapidly through the two Trp codons, covering regions 1 and 2 of the mRNA leader. Regions 3 and 4 pair to form the terminator hairpin followed by a poly-U sequence, causing RNA polymerase to terminate transcription before reaching trpE. Panel B (Low tryptophan): The ribosome stalls at the tandem Trp codons (in region 1), leaving region 2 free. Regions 2 and 3 pair to form the antiterminator hairpin, preventing the 3-4 terminator from forming. RNA polymerase reads through and transcribes the full operon (trpE through trpA).</image>

V. Comparison of Inducible and Repressible Operons

VI. Additional Prokaryotic Regulatory Mechanisms

Riboswitches

Riboswitches are RNA-based regulatory elements located in the 5' UTR of certain mRNAs that allow the mRNA itself to sense a small molecule metabolite directly, without any protein intermediary. When the metabolite binds, it induces a conformational change in the mRNA structure that can either form a terminator hairpin (blocking transcription) or sequester the Shine-Dalgarno sequence (blocking translation). Known riboswitches respond to metabolites including thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), S-adenosylmethionine (SAM), and various amino acids. Because riboswitches function without protein factors, they are thought to represent an ancient regulatory mechanism--possibly a relic of the RNA world, when RNA molecules performed both catalytic and regulatory functions.

Two-Component Signal Transduction Systems

Bacteria sense and respond to a wide range of external stimuli through two-component signal transduction systems. These consist of a sensor kinase--a membrane-spanning protein that detects an environmental signal and autophosphorylates on a histidine residue--and a response regulator that receives the phosphoryl group on an aspartate residue. Once phosphorylated, the response regulator functions as a transcription factor, binding DNA to activate or repress specific target genes. Two-component systems regulate diverse processes including chemotaxis, osmoregulation, and virulence gene expression.

Small Regulatory RNAs (sRNAs)

Prokaryotes also regulate gene expression post-transcriptionally through small regulatory RNAs (sRNAs)--short, non-coding RNA molecules of approximately 50-500 nucleotides. These sRNAs base-pair with target mRNAs, often with the assistance of the Hfq chaperone protein, and can block translation by occluding the ribosome binding site, promote mRNA degradation, or occasionally stabilize and enhance translation of their targets. In function, prokaryotic sRNAs are analogous to eukaryotic microRNAs.

VII. Global Regulation and Regulons

Beyond individual operons, bacteria coordinate the expression of large groups of genes in response to major environmental challenges. A regulon is a set of operons or genes scattered throughout the chromosome that are controlled by the same regulatory protein. The SOS response to DNA damage activates RecA protein, which stimulates self-cleavage of the LexA repressor, derepressing more than 40 genes involved in DNA repair, mutagenesis, and cell division arrest. The heat shock response to sudden temperature increases induces the alternative sigma factor sigma-32 (sigma-H), which directs RNA polymerase to the promoters of genes encoding molecular chaperones (DnaK, GroEL) and proteases. The stringent response to amino acid starvation triggers accumulation of the alarmone (p)ppGpp, which reprograms transcription by repressing rRNA and tRNA genes while activating amino acid biosynthesis genes.

These global responses rely heavily on alternative sigma factors--different sigma subunits that associate with the core RNA polymerase and redirect it to distinct sets of promoters. Sigma-70 directs transcription of housekeeping genes under standard conditions. Sigma-32 activates heat shock genes. Sigma-54 controls nitrogen metabolism genes. Sigma-S (sigma-38) governs stationary phase and general stress response genes. By switching sigma factors, bacteria can rapidly reprogram their entire transcriptional output to match changing environmental demands.

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