HMG - Week 12: Gene Expression in Bacteria LO

Why Bacteria Regulate Gene Expression

• Fundamental Principle: Bacteria must adapt rapidly to changes in their environment, unlike multicellular organisms that exist in more stable internal environments.

• Core Strategy: Bacteria only produce specific proteins when they are actually required. This efficient management allows the organism to:

  • Save energy: Protein synthesis is metabolically expensive.

  • Conserve resources: Raw materials (amino acids, nucleotides) are saved for essential functions.

  • Respond quickly: Gene regulation allows for rapid physiological shifts based on environmental stimuli.

  • Maximise growth and survival: By optimizing enzyme production, bacteria can outcompete others in a given niche.

• Conceptual Example (Lactose):

  • If lactose is absent: The bacteria has no need to produce the specialized enzymes required for lactose digestion.

  • If lactose is present: The bacteria triggers the production of enzymes required to metabolise lactose.

• Definition: This selective control of protein production is formally known as gene regulation.

Basic Features of E. coli as a Model Organism

• Classification: Escherichia coli (E. coli) is a Prokaryote.

• Key Biological Characteristics:

  • Unicellular organism.

  • Absent Nucleus: There is no nuclear envelope to sequester genetic material.

  • Lack of Organelles: No membrane-bound organelles (e.g., mitochondria, chloroplasts).

  • Nucleoid Region: The DNA is concentrated in a non-membrane-bound area called the nucleoid.

  • Circular Chromosome: Genetic material is organized into a single, circular loop.

  • Coupled Transcription and Translation: These two processes occur simultaneously in the cytoplasm.

Comparative Genetics: Prokaryotes vs Eukaryotes

• Nucleus: Absent in Prokaryotes; Present in Eukaryotes.

• DNA Structure: Circular in Prokaryotes; Linear in Eukaryotes.

• mRNA Processing: Minimal in Prokaryotes; Extensive in Eukaryotes (e.g., splicing, capping, tailing).

• Transcription Site: Cytoplasm in Prokaryotes; Nucleus in Eukaryotes.

• Translation Site: Cytoplasm in both Prokaryotes and Eukaryotes.

• Coupling: Yes in Prokaryotes; No in Eukaryotes.

• Important Exam Point: In bacteria, translation often begins before transcription of the mRNA molecule has even finished because there is no physical barrier (nucleus) separating the DNA from the ribosomes.

DNA and Transcription in Bacteria

• DNA Composition: Contains four nitrogenous bases:

  • Adenine ($A$)

  • Thymine ($T$)

  • Guanine ($G$)

  • Cytosine ($C$)

• Base Pairing Rules: $A$ pairs with $T$; $G$ pairs with $C$.

• The Blueprint: The specific sequence of these DNA bases provides the instructions for protein synthesis.

• Transcription Definition: The synthesis of messenger RNA (mRNA) from a DNA template.

• RNA Polymerase Enzyme: The primary enzyme responsible for reading the DNA template strand and producing a complementary mRNA strand.

• mRNA Bases: Adenine ($A$), Uracil ($U$), Guanine ($G$), and Cytosine ($C$). Note that Uracil replaces Thymine in RNA.

• Structure of E. coli RNA Polymerase: The enzyme complex (holoenzyme) is composed of several subunits:

  • $2 \, \alpha$ (alpha) subunits

  • $1 \, \beta$ (beta) subunit

  • $1 \, \beta'$ (beta-prime) subunit

  • $1 \, \omega$ (omega) subunit

  • Sigma ($\sigma$) factor: Required for the holoenzyme to specifically recognize and bind to promoters.

Translation and the Genetic Code

• Translation Definition: The synthesis of proteins from the mRNA sequence.

• Bacterial Ribosome Structure: Known as the $70S$ ribosome, it is composed of:

  • $30S$ small subunit

  • $50S$ large subunit

• Ribosome Function: Reads mRNA codons and links amino acids together to form polypeptide chains.

• The Codon: A sequence of three nucleotides that specifies a particular amino acid.

  • $AUG$: Methionine (also serves as the Start codon).

  • $UUU$: Phenylalanine.

  • $GCU$: Alanine.

• Degeneracy of the Genetic Code: Multiple different codons can code for the same amino acid.

  • Example (Leucine): Coded for by six different codons: $UUA$, $UUG$, $CUU$, $CUC$, $CUA$, and $CUG$.

The Operon Concept

• Definition: An operon is a cluster of genes controlled by a single promoter and transcribed together as one multi-genic mRNA molecule.

• Functional Components of an Operon:

  1. Regulatory Gene: A gene that produces a regulatory protein (like a repressor).

  2. Promoter: The specific DNA sequence where RNA polymerase binds to initiate transcription.

  3. Operator: The DNA sequence that serves as the binding site for repressor proteins; it acts as the physical switch for the operon.

  4. Structural Genes: The specific sequence of genes that encode the proteins required for a metabolic pathway.

The Lac Operon: Context and Discoveries

• Discovery: Discovered by François Jacob and Jacques Monod; it is the definitive example of bacterial gene regulation.

• Function: Enables E. coli to utilize Lactose as an alternative energy source when glucose is scarce.

• Lactose Properties: A disaccharide composed of Glucose and Galactose.

• Metabolic Requirements: Lactose cannot be used directly by the cell. It must undergo two processes:

  1. Transport: Moving lactose into the cell across the membrane.

  2. Hydrolysis: Breaking lactose down into glucose and galactose.

Structural Genes of the Lac Operon

• $lacZ$:

  • Encodes the enzyme $\beta$-Galactosidase.

  • Function 1: Cleaves lactose into glucose and galactose.

  • Function 2: Converts a small portion of lactose into allolactose, which serves as the inducer molecule.

• $lacY$:

  • Encodes Lactose Permease.

  • Function: A membrane-bound transport protein that pumps lactose into the bacterial cell.

• $lacA$:

  • Encodes Transacetylase.

  • Function: Its role is not fully established, but it is theorized to detoxify certain harmful byproducts.

Regulatory Elements of the Lac Operon

• Promoter ($lacP$): The binding site for RNA polymerase to begin transcription.

• Operator ($lacO$): The binding site for the lactose repressor protein. It is approximately $22$ base pairs long and is located adjacent to the promoter. It acts as the molecular "ON/OFF" switch.

• $lacI$ Gene: Located upstream of the structural genes and the promoter. It produces the Lac Repressor Protein.

• Jacob and Monod Model: Proposed that gene expression is regulated specifically by proteins binding to specific sequences of DNA. This became the foundation for modern gene regulation theory.

Negative Control of the Lac Operon

• Mechanism: Control is exerted by the action of a repressor protein preventing expression.

• State: Lactose is ABSENT:

  1. The $lacI$ gene constitutively produces the repressor protein.

  2. The repressor protein (a tetramer consisting of four subunits) binds to the operator ($lacO$).

  3. The physical presence of the repressor blocks RNA polymerase from moving past the promoter into the structural genes.

  4. No transcription occurs; no lac proteins are produced.

  5. Result: The Operon is OFF to prevent wasting energy.

• State: Lactose is PRESENT:

  1. Lactose enters the cell (via a small number of existing permease proteins).

  2. Some lactose is converted into allolactose (the true inducer molecule).

  3. Allolactose binds to the lac repressor protein.

  4. The repressor undergoes a conformational change (changes shape).

  5. The altered repressor can no longer bind to the operator.

  6. The operator becomes free, allowing RNA polymerase to transcribe the genes.

  7. $lacZ$, $lacY$, and $lacA$ proteins are produced.

  8. Result: The Operon is ON.

Genetic Mutations in the Lac Operon

• $lacI^+$ (Normal): Produces functional repressor; regulation is normal.

• $lacI^-$ Mutation:

  • Produces a defective repressor protein that cannot bind to the operator.

  • Result: The operon is permanently ON (constitutive expression) regardless of lactose presence.

  • Exam Phrase: $lacI^-$ mutations act in trans because the repressor protein is a diffusible molecule that can move throughout the cytoplasm to find an operator.

• $lacO^c$ (Operator Constitutive) Mutation:

  • A mutation in the DNA sequence of the operator itself.

  • The repressor protein cannot recognize or bind to the mutated DNA.

  • Result: The operon is permanently ON.

  • Cis-Acting: Unlike $lacI$, $lacO^c$ mutations act in cis, meaning they only affect the genes on the same physical strand of DNA where the mutation occurs.

Positive Control and Glucose Regulation

• Glucose Preference: E. coli prefers to use glucose as its primary carbon source. It will only fully activate the lac operon if glucose is unavailable.

• Catabolite Activator Protein (CAP): Also known as the CAP-cAMP system.

• Low Glucose Scenario:

  1. Intracellular levels of cyclic AMP (cAMP) increase.

  2. cAMP binds to CAP (Catabolite Activator Protein).

  3. The CAP-cAMP complex binds to a site near the promoter.

  4. This binding facilitates and increases the affinity of RNA polymerase for the promoter.

  5. Transcription increases significantly.

• High Glucose Scenario:

  1. cAMP levels decrease.

  2. CAP cannot bind to the DNA without cAMP.

  3. RNA polymerase binding is weak and inefficient.

  4. Transcription is greatly reduced, even if lactose is present.

• Condition for Maximum Expression: Requires Lactose to be present AND Glucose to be absent.

Summary: Operational States of the Lac Operon

High-Yield Exam Summary

• Inducible Operon: The lac operon is naturally "OFF" and must be induced to turn "ON."

• $lacZ$: $\beta$-galactosidase.

• $lacY$: Permease.

• $lacA$: Transacetylase.

• $lacI$: Encodes the repressor protein.

• Operator: Binding site for the repressor.

• Inducer: Allolactose (derived from lactose).

• $lacI^-$ and $lacO^c$: Both lead to constitutive (continuous) expression.

• Max Transcription Context: Lactose must be present to remove the repressor, and glucose must be absent to allow CAP-cAMP activation.