WEEK 8-Lac Operon, Mutations, Mutagens

The Lac Operon

• First operon discovered in E. coli

• Encodes enzymes for lactose metabolism

• Polycistronic mRNA with 3 genes:

  • lacZ: Encodes B-galactosidase (LacZ), breaks lactose into glucose and galactose

  • lacY: Encodes lactose permease (LacY), transports lactose into the cell

  • lacA: Encodes B-galactosidase transacetylase (LacA)

LacY (Permease) and LacZ (B-galactosidase)

• LacY allows lactose entry into the cell

• LacZ converts lactose into glucose and galactose

• Induced in gram-negative cells when lactose is present

Control of Gene Expression in Bacteria

• Sigma factors control multiple genes

• Gene expression controlled by repressors and inducers

• Small molecules can activate gene expression

Regulation of the Lac Operon

• Highly regulated; only transcribed when lactose is present

• Turned on in presence of lactose, turned off without lactose

• LacI (repressor) binds operator and blocks transcription in the absence of lactose

• LacI binding prevents RNA polymerase from initiating transcription

  

Repressor (LacI)

• Protein that prevents gene expression by binding the operator

• Blocks RNA polymerase from initiating transcription

• Bends DNA to prevent RNA polymerase access to lac promoter

Lac Operon in the Presence of Lactose

• Low or absent glucose with lactose induces operon

• Modified lactose binds repressor protein, changing its shape

• Repressor cannot bind the operator, allowing low-level transcription

• Inducer: substrate that induces expression of genes for its metabolism

Apoinducer and CAP

• Apoinducer: Protein that enhances gene expression

• Catabolite Activator Protein (CAP) binds promoter with cAMP

• cAMP (cyclic AMP) levels vary with nutrient availability:

  • Low glucose → high cAMP (forms CAP-cAMP complex)

  • High glucose → low cAMP (no CAP-cAMP complex)

Lac Operon Control

• Gene expression can be controlled by:

  • Repressors and inducers

  • Small molecule activation

• Two systems work together in Lac operon activation:

  • Inducer control: Lactose binds to repressor, disabling it to allow transcription

  • Small molecule activation: cAMP binds to CAP, enhancing transcription

Role of cAMP in Lac Operon

• cAMP levels inversely related to glucose levels

• Low glucose = high cAMP (CAP-cAMP enhances transcription)

• High glucose = low cAMP (no enhancement of transcription)

Catabolite Repression and Diauxic Growth

• Catabolite repression causes bacteria to preferentially use glucose

• Diauxic growth: Glucose metabolism first, followed by lactose metabolism

X-gal as a Substrate for B-gal

• X-gal: alternative substrate for B-galactosidase

• Creates a blue-colored product indicating B-gal activity

The trp Operon (Repressible Operon)

• Encodes enzymes for tryptophan biosynthesis

• Repressor protein binds tryptophan to inactivate the operon

• High tryptophan levels inhibit the operon, preventing excess

Mutations

• Heritable changes in the DNA sequence

• Effects on the organism:

  • Almost always deleterious

  • Sometimes neutral (no effect)

  • Rarely beneficial (new property)

  • When you still see bacteria in zone of inhibition it is because those colonies have undergone a mutation that is resistant.

Heritability of Mutations

• Changes in DNA sequence become heritable through replication

• Mutant: cell line inheriting a specific mutation

Points Mutations

• Single nucleotide base pair mutation

• Types:

  • Substitution: e.g., "THE CAT ATE ELK" -> "THE RAT ATE ELK"

  • Insertion: e.g., "THE CAT ATE ELK" -> "TRH ECA TAT EEL K"

  • Deletion: e.g., "THE CAT ATE ELK" -> "TEC ATA TEE LK"

Genotype vs Phenotype

• Genotype: DNA sequence

• Phenotype: Observable trait (e.g., protein function)

• Wild-type (Genotype)-> mRNA-> Protein (Phenotype)

Silent Mutations

• Genotype changes, phenotype unchanged

• DNA is mutated -> genotype changes

• Protein is NOT changed-> phenotype doesn't change

• Example: CAA -> CAG (same amino acid)

Missense Mutation

• Amino acid substitution

• May affect phenotype, depending on location

• DNA is mutated-> genotype changes

• Protein is changed -> phenotype changes may occur depending on location

• Example: CAA -> CAT

Nonsense Mutations

• Amino acid changed to stop codon

• Typically causes loss of function

• DNA mutated → genotype changes

• Protein changed=loss of function in addition to phenotypic change

• Example: CAG -> TAG

Physical Mutagens

• UV light: Causes C=C or T=T dimers

• X-rays: Break DNA backbone bonds

Chemical Mutagens

• Nucleotide Analogs: Resemble nucleotides, cause replication errors and mispairing

• Nucleotide-Altering Chemicals: Alter base structure to a different base or base analog

• Framsift Mutagens

Frameshift Mutagens

• Insert between DNA bases, causing insertions or deletions during replication

DNA Repair Mechanisms

Direct Repair, Single Strand Repair, Error-prone repair

Direct Repair

• Small damage on one strand

• Examples:

  • Base-Excision Repair: Removes mutated base, replaces it

  • Light Repair: Photolyase enzyme fixes C=C or T=T dimers

Single Strand Repair

• Nucleotide Excision Repair: Cuts out damaged section, uses template for repair

• Mismatch Repair: Removes mismatched bases

Error-Prone Repair

• SOS Response: Fills gaps with random sequences as a last resort

Ames Tests

Used to identify mutagens

• Uses Salmonella bacteria to test if a substance is a mutagen

• Presence of colonies indicates mutagenic potential