Week 3 S - Gene Regulation in Prokaryotes

Catabolite Repression

• Diauxic Growth of E. coli: Illustrates the preferential use of glucose over lactose.

  • When both glucose and lactose are present, E. coli will use glucose first.

  • Once glucose is depleted, there is a lag phase before growth resumes on lactose.

  • This results in a biphasic growth curve.

  • The activity of β-galactosidase, an enzyme required for lactose metabolism, increases only after glucose is used up.

Why is Gene Regulation Important to Bacteria?

• Adaptation: Allows bacteria to adapt to changes in their environment.

• Resource Optimization: Ensures optimal utilization of cellular resources.

The Operon Model

• Discovery: Proposed by Jacques Monod and Francoise Jacob in 1961.

• Trans-Acting Regulatory Factor: A regulatory protein that can affect genes located anywhere in the genome.

• Cis-Acting Regulatory Element: A DNA sequence (operator or promoter) located adjacent to the regulatory target gene.

Gene Control Mechanisms

• Negative Control: A trans-acting repressor prevents/reduces gene expression by binding to a cis-element.

• Positive Control: A trans-acting regulator/activator increases gene expression by binding to a cis-element.

• Gene Induction: When a gene is switched on by a signal.

• Gene Repression: When a gene is switched off by a signal.

• Co-effector: Small molecules that cause induction/repression.

• Operon: Multiple genes controlled by one promoter/operator, resulting in a single transcript.

The lac Operon

• Components: Includes:

  • lacI: Gene for the repressor protein.

  • o/p: Operator/promoter region.

  • lacZ: Gene for β-galactosidase.

  • lacY: Gene for permease.

  • lacA: Gene for transacetylase.

• Transcription Start: Region where transcription begins.

• Structural Genes: lacZ, lacY, and lacA.

• Repressor: Encoded by lacI, a homotetramer of approximately 125 kDa (x4).

• β-galactosidase: A protein of about 30 kDa.

• Permease: An inner membrane protein of about 30 kDa.

lacZYA Promoter Region

• Key regions are numbered, indicating specific binding sites and regulatory elements.

lacZYA Control

• Induction à Expression: Process of switching on gene expression.

• Time Delay: There is a time delay in induction because:

  • Time is needed for transcription and translation to progress.

• Half-Life of lac Operon mRNA: Approximately 3 minutes.

  • Means that 50% of the mRNA degrades in 3 minutes.

  • Once transcription stops, expression quickly diminishes.

Induction by Lactose

• Allolactose: The direct inducer of the lac operon.

  • Isomer of lactose.

  • Generated as a side-reaction product of lactose by LacZ (β-galactosidase).

• Two Extra Steps Needed for Induction via Lactose:

  1. Uptake of lactose into the cell via LacY (permease).

  2. Reaction of lactose with β-Gal enzyme.

LacI (Lac Repressor)

• Subunit Structure: Homo-tetramer.

• Binding: Binds to the operator in the absence of the inducer.

• Effect of Inducer Binding: Loss of binding of LacI to the operator à induction of lac operon.

• Allosteric Control: Shape change upon inducer/allolactose binding causes loss of DNA binding.

  • Allolactose binds, causing a conformational change.

  • This leads to a loss of DNA binding affinity.

LacI – Subunit Structure

• N-terminus: Contains the DNA-binding domain with a helix-turn-helix motif and ‘hinge’.

• Core Domains: Involved in allolactose binding (dimerization) and tetramer formation.

• Mutations: Impacting:

  • DNA-binding domain.

  • Inducer-binding site.

  • Homo-dimer formation.

  • Tetramer formation.

Lac Operator

• Palindrome: An inverted repeat.

• Lac Operator: Yes, it consists of two 16 bp inverted repeats.

• Operator Half-Site: One of the inverted repeat sequences.

• Palindromicity: Reflects the structure of LacI.

• Effect of Missing Half-Site: Very poor LacI binding to DNA; very weak LacI repression (lac operon is constitutive).

The LacI Homo-tetramer

• Tetramer Formation: Allows binding to two operators simultaneously, increasing binding affinity and repression.

• Number of LacI Operators: Three in total: O1, O2, and O3.

• Effect of Loss of O2 and O3: X50 reduction in LacI repression.

Operator Binding and DNA Looping

• Operator Strengths: O1 (strong), O2 (weak), O3 (weak).

• DNA Looping: The LacI tetramer binds to two operators that are hundreds of base pairs apart, forming a DNA loop.

Second Layer of lac Operon Control

• Effect of Glucose: Causes repression (catabolite repression) of the lac operon.

• Other Genes Affected: Yes, <20 additional operons are also subject to catabolite repression.

• Purpose: Glucose is the preferred substrate/better energy source for E. coli.

• Signal Molecule: cAMP (cyclic AMP).

• Regulatory Protein: CRP – cAMP Receptor Protein.

CRP

• Role: Activates gene expression.

• Co-effector: cAMP.

• cAMP Generation: From ATP by adenylate cyclase.

• cAMP Levels: Adenylate cyclase activity is inhibited by glucose.

• Relationship Between cAMP and Glucose: Reciprocal; high glucose leads to low cAMP and vice versa.

CRP Function

• Subunit Structure: Homodimer.

• Binding Components: Each CRP can bind three different components: cAMP, DNA binding site, and RNA polymerase.

• DNA-Binding Site Features: Palindrome of 10 bp; total binding site covers 22 bp; 6 bp spacer between the two half sites.

• Hyphenated Palindrome: The CRP DNA-binding site is described as a ‘hyphenated’ palindrome because of the 6 bp spacer between the two half sites.

Similarities and Differences – LacI v Crp

• LacI:

  • Repressor.

  • Tetramer.

  • Does not interact with RNA pol.

  • HTH DNA-binding domain.

  • Binds to a palindromic DNA sequence.

• Crp:

  • Activator.

  • Dimer.

  • Interacts with RNA pol.

  • HTH DNA-binding domain (SAME).

  • Binds to a palindromic DNA sequence (SAME).

CRP – Mutation Consequences

• Inactivation of Adenylate Cyclase: Permanent catabolite repression.

  • Compensation: Add cAMP to the medium.

• Unregulated Adenylate Cyclase Activity: Loss of catabolite repression.

• Inactivation CRP’s Binding to RNA Polymerase: Failure to activate expression – permanent catabolite repression effect (even without glucose).