145lec13F21

Learning Objectives

• Understand the concepts of repression and induction in gene regulation.

• Explore specific operons, including lac operon, arabinose operon, and trp operon, to illustrate the mechanisms of gene expression regulation.

Repression

• Definition: Repression is a regulatory mechanism that inhibits gene expression, effectively turning off the gene when it is not needed. This process ensures energy efficiency and proper cellular function. This is often achieved through the binding of a repressor protein to the operator region of the operon, blocking RNA polymerase from transcribing the downstream genes.

• Mechanism: A regulator protein known as a repressor binds to the DNA at specific sites, namely the operator region, preventing the transcription of genes into RNA by RNA polymerase (RNAP).

  • The repressor may require a co-factor, which is often a product of the structural gene that it regulates, enhancing its ability to bind to DNA and exert repression.

Enhancers/Activators

• Function: Enhancers are DNA sequences that, when bound by specific proteins called activators, promote the transcription of associated genes at the promoter by facilitating the recruitment of RNA polymerase.

  • Promoter Activation: Enhancers help make the promoter region more accessible to RNAP or may enhance the binding of the sigma factor, increasing the likelihood of transcription initiation.

Escherichia coli

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• Transformation Methods: Techniques such as transformation with strains like J1-BTH101 on LB agar plates supplemented with antibiotics (ampicillin and kanamycin) are essential for introducing plasmids into bacterial cells to study gene expression.

• Detection Assays: Various assays, including colorimetric assays and the B-galactosidase assay, are widely used to detect protein-protein interactions and gene expression levels, providing insights into the functionality of genetic constructs.

Induction

• Definition: Induction refers to the activation of genes in response to a specific inducer, often a substrate that the operon is designed to metabolize. This process contrasts with repression and plays a critical role in metabolic regulation.

• Inducer Role: The inducer typically serves as the substrate for the metabolic pathway encoded by the genes of the operon, allowing cells to conserve resources by expressing only the necessary genes in the presence of that substrate.

• Operon Concept: An operon is defined as a set of contiguous genes within the genome that are coordinately regulated, enabling the simultaneous expression of functionally related proteins.

  • Repressors and inducers work in tandem to finely tune gene expression and ensure proper metabolic responses.

The Lac Operon

• Components: The lac operon consists of structural genes lacZ, lacY, and lacA, alongside the regulatory gene lacI, and key regions such as the promoter and operator.

• Functionality:

  • In the absence of lactose, the repressor protein binds to the operator region of the lac operon, effectively preventing transcription of the downstream genes.

  • When lactose is present, it is converted to allolactose, which binds to the LacI repressor, causing it to release from the DNA and allowing transcription to proceed, facilitating the metabolism of lactose.

• Catabolite Repression: This phenomenon explains how the presence of glucose (the preferred substrate for E. coli) regulates the lac operon. The key regulatory protein, CAP (catabolite activator protein), which is produced from the crp gene, interacts with cyclic AMP (cAMP) levels that inversely correlate with glucose availability, thus modulating the transcription level of the lac operon in response to glucose presence.

Arabinose Operon

• Control Mechanisms: The arabinose operon employs both positive and negative control mechanisms to regulate gene expression.

• AraC Protein: This protein acts as a dual-function regulator; it serves as a repressor in the absence of arabinose by forming a repressor loop. However, when arabinose is present, it binds to AraC, causing a conformational change that allows transcription to proceed by displacing the repressor.

  • AraC also engages with cAMP-CAP to boost transcription rates further under conditions favorable for arabinose metabolism.

Repression of the Trp Operon

• Function: The trp operon is responsible for the biosynthesis of the amino acid tryptophan, a vital component for protein synthesis.

• Mechanism: When intracellular tryptophan concentrations are high, the tryptophan molecule enhances the binding of the repressor to the trp operon, effectively shutting down the synthesis pathway for unnecessary tryptophan, thereby conserving cellular resources.

• Transcription Attenuation: The leader region of the trp operon contains Trp codons that influence ribosome movement during translation. If tryptophan levels are sufficient, the ribosome proceeds unhindered, leading to the formation of a transcription-terminating secondary structure in mRNA, effectively halting further transcription and allowing regulation of tryptophan biosynthesis.