Prokaryotic Gene Regulation Overview

•Overview • Genetic Switches • Lac Operon – Lactose Induction • Adaptive Value • Lac Operon – Catabolite Repression – Response to environment • Expression of fatty acid desaturases in response to • Trp Operon – Attenuation lower temperatures – Development and Differentiation • House Keeping vs Luxury Genes/Proteins – Example Liver vs. Muscle

Lac Operon Gene Induction

• Inducer – Lactose • Beta Galactosidase • Lactose Permease • Transacetylase

• Analogue Inducer - IPTG

Lac Operon Components of Regulation

Cis elements – DNA sequences adjacent to gene that play a role in transciptional regulation – Lac P, Lac O

Trans Factors – Proteins that bind to cis elements and mediate regulation – Lac I (Lac Repressor), RNA Polymerase

Inducer - Lactose

1 Repressor Function Lactose Induction

Lactose binding Shifts equilibrium Lac Lac Repressor Repressor

High Affinity Low Affinity For operator for operator

Evidence for Model Partial Diploids

B-gal Activity B-gal Activity Mutation - lactose + lactose type Reg Diploid - lactose + lactose type Reg I+O+/F’I+ LacI- I+Oc/F’I+ Promoter I+O+/F’Is LacOc

LacIs

Multiple Lac Operators Lac Repressor Tetramer

Fold Repression

1300

1

18

400

700

2 Catabolite Repression Players in Catabolite Repression

• Lac Operon is inhibited by glucose even when lactose is present. Inhibition by cAMP CAP

glucose is called “catabolite repression” Phospho- diesterase Adenyl Glucose Cyclase • Adaptive significance – Advantages for bacteria to consumer “cheaper sugar” over more “expensive sugar” when both are available. CBS Promoter Lac Operon

non-consensus Promoter

cAMP Catabolite Repression

Adenyl Phospho- cyclase diesterase

Adenylate cyclase is inhibited by glucose Glucose present – cAMP concentration low Glucose absent – cAMP concentration high

Consider the following mutations Trp operon

• Constitutive Repression – always off –CAP – Adenylate Cyclase – CAP Binding Site –Promoter • Non Repressible – glucose doesn’t inhibit transcription –CAP – Adenylate Cyclase –Promoter – Phosphodiesterase

3 Attenuation

Leader Peptide Attenuation Region

Low Tryptophan Conditions 1. When tryptophan is low, charged trp tRNA’s are low. 2. This causes the ribosome to pause in attenuation region over UGG codons 3. The ribosome prevent the leader peptide from participating in secondary structure 4. This prevents the termination complex from forming.

High Tryptophan Conditions 1. When tryptophan is high there are abundant charged trp tRNA’s. 2. This allows the ribosomes to rapidly translate LP. 3. LP can participate in secondary structures. 4. Terminator structure forms

Consider effect of mutations in LP and A

4