regulation of gene expression by e. börje lindström this learning object has been funded by the...
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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)
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