Regulation of Gene Expression in Bacteria: The lac and trp Operons (CH 15)

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Regulation of Gene Expression in Bacteria

Chapter Concepts

Regulation of gene expression in bacteria is closely linked to the cell's metabolic needs. Bacterial genes encoding proteins with related functions are often organized in clusters called operons, allowing coordinated control. Transcription within operons can be inducible or repressible, and regulatory mechanisms are classified as exerting either positive or negative control. The end product of a metabolic pathway often induces or represses gene expression in that pathway.

Operon: A cluster of genes under coordinated control, including regulatory sequences.
Inducible system: Genes are expressed only in the presence of a specific inducer.
Repressible system: Genes are expressed unless repressed by a specific molecule.

Lactose Metabolism in E. coli: The Inducible lac Operon

Negative Control of the lac Operon

The lac operon in E. coli is a classic example of an inducible system regulated by negative control. Genes involved in lactose metabolism are expressed only when lactose is present and glucose is absent.

lacI gene: Encodes the repressor protein that binds the operator to block transcription.
Inducer (allolactose): Binds the repressor, causing it to release the operator and allowing transcription.
Negative control: Repressor binding prevents RNA polymerase from transcribing the structural genes.

Constitutive Mutations

Constitutive mutations disrupt normal regulation, causing continuous expression of the operon.

I- mutation: Mutation in the repressor gene prevents repressor binding to the operator, leading to constitutive synthesis of the lac operon.
Oc mutation: Mutation in the operator site prevents repressor binding, also resulting in constitutive expression.

lacIS Super-repressor Mutation

The lacIS mutation produces a repressor that cannot interact with the inducer (allolactose), keeping the operon permanently repressed and non-inducible.

Repressor always binds the operator, blocking transcription regardless of lactose presence.
Introduction of a wild-type lacI+ gene does not relieve repression.

Isolation of the Repressor

The nature of the lac repressor was clarified in 1966 when G. and Muller-Hill isolated it in purified form. Experiments using equilibrium dialysis with radioactive IPTG demonstrated that the repressor is a protein, not an RNA molecule.

Allosteric protein: Changes conformation upon binding the inducer.
Equilibrium dialysis: Showed repressor does not diffuse, confirming its protein nature.

Genetic Proof of the Operon Model

Three key predictions validated the operon model:

lacI gene: Produces a diffusible, trans-acting product.
Operator (O): Involved in regulation, has no protein product.
Operator adjacency: Must be adjacent to structural genes to regulate them.

Partially diploid bacteria (merozygotes) created using F' plasmids were used to test these predictions.

Merozygotes

A merozygote is a bacterial cell that is diploid for certain genes due to acquisition of an F' plasmid. Merozygotes were used to analyze gene activity in the presence or absence of lactose.

Genotype	Lactose Present	Lactose Absent
I+O+Z+/I-O+Z+	+	-
I+OcZ+/I+O+Z+	+	+
ISO+Z+/I+O+Z+	-	-
I-O+Z+/I+O+Z+	+	-
I+O+Z-/I+O+Z+	+	-
I+O+Z+/I+O+Z-	+	-

Additional info: Table entries inferred from standard lac operon analysis.

The Catabolite-Activating Protein (CAP) and Positive Control

CAP and Catabolite Repression

The Catabolite-Activating Protein (CAP) exerts positive control over the lac operon. When glucose is absent and lactose is present, CAP binds to the CAP-binding site, facilitating RNA polymerase binding and promoting transcription.

CAP: Activator protein that enhances transcription in the absence of glucose.
Catabolite repression: Presence of glucose inhibits lac operon expression.

Role of cAMP

CAP must be bound to cyclic Adenosine Monophosphate (cAMP) to bind the promoter region. Glucose inhibits Adenylyl Cyclase, reducing cAMP levels and preventing CAP from binding.

Adenylyl Cyclase: Converts ATP to cAMP.
cAMP-CAP complex: Required for positive control of the lac operon.

Combined Regulation

Transcription of the lac operon is determined by both positive (CAP-cAMP) and negative (repressor) regulation.

High transcription: Lactose present, glucose absent (CAP-cAMP active, repressor inactive).
Low/no transcription: Lactose absent or glucose present (repressor active or CAP-cAMP inactive).

Mechanisms of Transcriptional Control

Negative Control

Inducer effect: Inducer binds repressor, preventing it from binding the operator, allowing transcription.
Corepressor effect: Corepressor binds repressor, enabling it to bind the operator and block transcription.

Positive Control

Allosteric effector: Effector binds activator protein, allowing it to bind DNA and promote transcription.
Allosteric inhibitor: Inhibitor prevents activator protein from binding DNA, blocking transcription.

The Tryptophan (trp) Operon: A Repressible System

Organization and Function

The trp operon in E. coli consists of five contiguous genes (trpE, trpD, trpC, trpB, trpA) encoding enzymes for tryptophan synthesis. The operon is regulated by a repressible system.

trpR gene: Encodes the repressor protein.
trpP: Promoter region, binding site for RNA polymerase.
trpO: Operator region, bound by the repressor.

Regulation by Tryptophan

Absence of tryptophan: Repressor cannot bind operator; transcription occurs.
Presence of tryptophan: Tryptophan acts as a corepressor, binds repressor, enabling it to bind operator and block transcription.

Leader Sequence and Attenuation

The trp operon contains a Leader Sequence (L) with a regulatory site called the Attenuator (A). Attenuation is a secondary regulatory mechanism that fine-tunes gene expression.

Attenuation: When the operon is repressed, transcription initiation still occurs at low levels, but is prematurely terminated.
Hairpin structures: mRNA can fold into stem-loop structures (hairpins) that determine whether transcription continues or terminates.

Mechanism of Attenuation

Low tryptophan: Ribosome stalls at Trp codons, mRNA forms antiterminator hairpin (regions 2-3), allowing transcription.
High tryptophan: mRNA forms terminator hairpin (regions 3-4), terminating transcription prematurely.

Attenuation in Other Operons

Attenuation is also found in operons for threonine, histidine, leucine, and phenylalanine biosynthesis.

Summary Table: Comparison of lac and trp Operons

Feature	lac Operon	trp Operon
Type of Regulation	Inducible	Repressible
Regulatory Molecule	Inducer (allolactose)	Corepressor (tryptophan)
Control Mechanism	Negative and Positive	Negative (plus attenuation)
Gene Products	Lactose metabolism enzymes	Tryptophan biosynthesis enzymes
Additional Regulation	CAP-cAMP (positive control)	Attenuation via leader sequence