genetics ch 11 anderson fordham

Lactose metabolism in E. coli

Controlled by an inducible system

Allolactose

Functions as the inducer of the lac operon

lac structural genes

Z, Y, and A

Function of lac Z

Encodes β-galactosidase which cleaves lactose into glucose and galactose

Function of lac Y

Encodes permease which transports lactose into the cell

Function of lac A

Encodes transacetylase involved in lactose metabolism

lac operon transcription

Structural genes transcribed together into a single mRNA

lac operon regulatory region

Contains promoter (P) and operator (O) upstream of structural genes

I gene

Lac repressor gene encoding a diffusible protein

Promoter (P)

DNA site where RNA polymerase binds to initiate transcription

Operator (O)

DNA site where repressor binds to block transcription

Induction mechanism

Allolactose binds repressor → repressor changes shape → cannot bind operator → transcription proceeds

lac operon "off" state

Lactose absent → repressor binds operator → transcription blocked

lac operon "on" state

Lactose present → allolactose binds repressor → repressor released → transcription proceeds

Jacob and Monod (1960)

Proposed operon model where a group of genes is regulated and expressed together

Studying gene regulation

Biochemical assay, conditions altering expression, and mutations that affect expression

Synthetic inducers

Used to study lac operon control mechanisms, non-hydrolyzable by β-galactosidase

F′ factors

Used to create partially diploid bacteria for genetic analysis of lac mutations

Z⁻ and Y⁻ alleles

Recessive to wild-type Z⁺ and Y⁺

Oᶜ mutation

Operator-constitutive mutation causing continuous expression regardless of inducer

Operator function

Cis-acting site affecting only genes on the same DNA molecule

Repressor function

Trans-acting protein that can diffuse and regulate both operons in the same cell

Iˢ (superrepressor) mutation

Repressor permanently active, cannot bind inducer, dominant to I⁺

Allosteric regulation in lac operon

Repressor changes conformation when bound to allolactose

Promoter sequence

Conserved -10 and -35 boxes critical for RNA polymerase binding

Mutations in promoter

Reduce transcription efficiency

CAP (Catabolite Activator Protein)

Activates lac operon transcription in response to cAMP

cAMP levels

High when glucose is low, low when glucose is high

CAP-cAMP complex

Binds near promoter, bends DNA, facilitates RNA polymerase binding

Catabolite repression

Lac operon remains off in presence of glucose even if lactose is present

DNA binding symmetry

Lac repressor tetramer binds operator, CAP dimer binds activator site

Lac operon regulation summary

Repression: active repressor binds operator → off, Induction: inducer binds repressor → on, Activation: CAP-cAMP binds → on, No activation: CAP inactive → off

Arabinose operon

Exhibits dual positive and negative regulation

AraC protein

Functions as both activator and repressor depending on arabinose presence

araC gene

Encodes AraC protein

Arabinose present

AraC-arabinose complex binds araI → transcription activated

Arabinose absent

AraC binds araI and araO → DNA loop forms → transcription repressed

CAP-cAMP in arabinose operon

Binds adjacent site and activates transcription with AraC when arabinose is present and glucose absent

trp operon

Contains trpE, trpD, trpC, trpB, trpA for tryptophan synthesis

trp operon regulation

Repression via repressor binding operator, Attenuation via leader sequence secondary structures

Attenuation

Decreases mRNA production when tryptophan is abundant

Leader sequence in trp operon

Encodes leader peptide sensing tryptophan levels, forms stem-loop structures

High tryptophan

Terminator stem-loop forms → transcription stops

Low tryptophan

Ribosome stalls at trp codons → anti-terminator forms → transcription continues

Attenuation mechanism

Relies on complementary base pairing in leader mRNA

Other amino acid operons

Use similar attenuation systems with leader peptides containing codons for their specific amino acid