Regulation of Gene expression

by

E. Börje Lindström

This learning object has been funded by the European Commissions FP6 BioMinE project

Introduction• Biosynthetic reactions consume energy: Sophisticated control

mechanisms in bacteria

• Available energy is limited in Nature:

Production of as much cell material per energy as possible

• The environment is important: - the nutrient in the medium is used first

- rapid and drastic changes in the nutrients

- reversible control reactions needed

• Two types of model systems:

- Biosynthetic

- Catabolic

Biosynthetic reactionsTryptophan is chosen as a model system:

- Tryptophan is an essential amino acid

- Tryptophan is missing in some plant proteins

- of industrial importance

• The bacterial cells are controlling the biosynthesis of tryptophan in three ways:

- feedback inhibition

- end product repression

- attenuation

Biosynthetic reactions, cont.• Feedback inhibition:

- The biosynthesis of tryptophan occurs in several steps:

Chorismate + glutamine antranilic acid B C D tryptophanE5E4E3E2E1

Mechanism: - enzyme E1 (the first enzyme) is an allosteric protein with

- a binding site for for the substrate

- a binding site for the effectors (inhibitor = try)

• E1 + try [E1-try]-complex that is inactive

• the complete biosynthesis of try is stopped

Biosynthetic reactions, cont.

• End product repression (EPR):

- In spite of ’end product inhibition’

- loss of energy due to enzymes E2-E5 are still synthesized

- another regulation is needed

- end product repression

Biosynthetic reactions, cont.Mechanism:

P O att E1 E3E2 E5E4

P = promoter;

O = operator

att = attenuator

E1 – E5 = structural genes for the enzymes E1-E5.

• RNA polymerase binds to P Initiation of mRNA synthesis

• The repressor binds to O Blocks the RNA polymerase movement

• The repressor is an allosteric protein

- inactive without tryptophan (does not bind to the operator)

• tryptophan acts as co-repressor -binds to the repressor

- makes the repressor active

Biosynthetic reactions, cont.

• Attenuator region: - barrier for the RNA polymerase

1) + try the polymerase removed from the DNA

2) - try the polymerase continues into the structural genes

• EPR inhibits all enzymes in tryptophan biosynthesis

save energy

- however, a slow total inhibition – does not effect already existing enzymes- high specificity – only the tryptophan operon is effected

Biosynthetic reactions, cont.

Biosynthetic reactions, cont.

Biosynthetic reactions, cont.

Biosynthetic reactions, cont.

Catabolic reactions• Catabolic systems are inducible

• Model system – lactose operon in E. coli

• The inducer is the available carbon/energy source

R P O lacAlacYlacZ

• Where:

- gene R : repressor protein – active without the inducer

- blocks mRNA polymerase

- gene lacZ : -galactosidase – splits lactose into glycose + galactose

- gene lacY: permease – transport lactose into the cell

- no attenuator sequence in catabolic systems

Catabolic reactions, cont.

• Mechanism:

+ lactose: - transported into the cell transformed into allo-lactose (inducer)

- allo-lactose + repressor [allo-lactose-repressor]- complex inactive

- RNA polymerase starts transcription of lactose operon

- -galactosidase is produced break down of lactose

- lactose: -[allo-lactose-repressor]- complex disintegrate

- the repressor binds to O and blocks further transcription of the operon

Catabolic reactions, cont.

Catabolic reactions, cont.

Catabolic repression (glucose-effect)

• Works in bacteria and other prokaryotes (here in E. Coli K12)

• Diauxi: - growth on two energy sources glucose + lactose

- two-step growth curve

Log OD

time

glucose

lactose

Growth on lactose

Growth on glucose

Catabolic repression (glucose-effect)

• Mechanism:

-cAMP an important substance

- required for initiation of transcription of many inducible systems

- global regulation

- glucose present [cAMP] (decreases)

- CAP (katabolite activator protein) an allosteric protein

- [cAMP-CAP]-complex binds to the promoter promotes transcription

-production of -galactosidase -1) lactose present

- 2) [cAMP-CAP]-complex present

Catabolic repression (glucose-effect), cont.

• + glucose:

- no [cAMP-CAP]-complex

- no transcription of lactose operon

- no -galactosidase production

• - glucose:

- [cAMP-CAP]-complex present

- transcription of lactose operon

- -galactosidase production

- brake down of lactose

Catabolic repression (glucose-effect), cont.

• Conclusions:

- Katabolite repression – a very useful function in bacteria

- forces the bacteria to use the best energy source first

Other types of Regulations• Constitutive systems:

- Enzymes that are needed during all types of growth

- e.g. those involved in glycolysis

- no regulation

- always present

• mRNA: - Unstable

- half-life ~ 2 min sub-units

- new mRNA

• polycistronic mRNA - one operator for several genes

• monocistronic mRNA - one operator per gene (in eukaryotes)