Regulation of Gene Expression

Regulation of Prokaryote Gene Expression

Learning Outcomes

• Describe E. coli lactose operon: gene names and protein products.
• Outline transcriptional regulation by lac repressor protein and catabolite activator protein (CAP).
• Explain effects of mutations in the lac operon.
• Describe E. coli arabinose operon: gene names and protein products.
• Understand regulation of arabinose operon with/without arabinose.
• Prepare for practical 2.
• Answer Workshop PSW5 questions.

Key Prior Information

• Knowledge of transcription and translation is crucial.
• Recommended reading: Klug, W.S., et al. "Essentials of Genetics," Chapters 12 & 13.
  • Chp 12: Genetic code and transcription.
  • Chp 13: Translation and proteins.

Gene Expression Control in Prokaryotes

Constitutive Gene Expression

• Housekeeping genes are always expressed for routine tasks.
• Example: Genes for amino acids, transcription (RNA polymerase), translation, tRNA, and rRNA.

Regulated Gene Expression

• Genes transcribed in response to environmental changes.
• Examples:
  1. Nutrient-responsive genes.
  2. Heat shock proteins.
• Focus on regulation in response to lactose and arabinose.

Control of Transcription

• Positive Control: Activator protein binds to DNA and initiates transcription.
• Negative Control: Repressor protein binds to DNA, preventing transcription.

Escherichia coli Lactose Operon

What is an Operon?

• Clusters of genes with coordinated transcriptional regulation by a shared regulatory region.
• Common in bacterial genomes, participating in similar metabolic/biosynthetic pathways.

Lac Operon Structure

• Regulatory region and three protein coding genes: lacZ, lacY, lacA.
  • lacZ: Encodes β-galactosidase.
  • lacY: Encodes lactose permease.
  • lacA: Encodes transacetylase.
• Regulatory components:
  • lac Operon Promoter: Binds RNA polymerase.
  • Operator (lacO): Binds lac repressor protein.
  • CAP binding site: Binds CAP-cAMP.

Lac Operon Control Mechanisms

• No Lactose (Glucose Available): Low expression; repressor binds to operator.
• Lactose Available (No Glucose):
  • Allolactose (from lactose) binds to repressor, preventing it from binding to operator.
  • RNA polymerase transcribes operon mRNA.

Additional Positive Regulation

• CAP-cAMP: Stimulates transcription when glucose is low.
• cAMP binds to CAP, which then binds to CAP binding site, enhancing transcription.

Mutations Affecting Lac Operon

1. lacI Mutation: Repressor can't bind; operon constitutively expressed.
2. lacOc Mutation: Mutated operator sequence; repressor can't bind; operon constitutively expressed.
3. lacIS Mutation: Super repressor, can't bind allolactose; operon always repressed.

Escherichia coli Arabinose Operon

Arabinose as a Nutrient Source

• Released in intestines from plant material; not absorbed but used by gut E. coli if glucose is absent.

Structure and Regulation

• Produces enzymes: araB, araA, araD for arabinose breakdown.
• AraC protein: Single regulatory protein with both positive and negative roles.
• No Arabinose: araC monomers bind to araI and araO2, form DNA loop, preventing RNA polymerase access.
• With Arabinose: Arabinose binding breaks loop, RNA polymerase binds, transcription initiated.
• CAP-cAMP helps enhance RNA polymerase binding when glucose is absent.

Expression Vectors

• pGLO plasmid utilizes the arabinose operon promoter.
• Arabinose presence triggers expression of constructs like GFP.

Additional Reading

• Schleif, R. "Regulation of the L-arabinose operon of Escherichia coli." Trends in Genetics.
• Workshop Questions: Address parts A, B, and C, focusing on gene expression regulation.