Transcript Regulation of Gene expression

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:
• Available energy is limited in
Nature:
• The environment is important:
 Sophisticated control
mechanisms in bacteria
 Production of as much cell
material per energy as possible
- 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 reactions
Tryptophan 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:
E1 antranilic acid E2
E3 C E4
E5 tryptophan
Chorismate + glutamine 
B
D
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
E2
E3
E4
E5
E1 – E5 = structural genes for the
enzymes E1-E5.
P = promoter;
O = operator
att = attenuator
 Initiation of mRNA synthesis
• RNA polymerase binds to P
• 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
• The repressor binds to O
 Blocks the RNA polymerase
movement
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
• The inducer is the available carbon/energy source
• Model system – lactose operon in E. coli
R
P
O
lacZ
lacY
lacA
• Where:
- gene R : repressor protein – active without the inducer
-  blocks mRNA polymerase
- gene lacZ : b-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
-  b-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 (glucoseeffect)
• 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
Growth on lactose
lactose
Growth on
glucose
time
glucose
Catabolic repression (glucoseeffect)
• 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 b-galactosidase  -1) lactose present
- 2) [cAMP-CAP]-complex present
Catabolic repression (glucoseeffect), cont.
• + glucose:
- no [cAMP-CAP]-complex 
- no transcription of lactose operon
- no b-galactosidase production
• - glucose:
- [cAMP-CAP]-complex present 
- transcription of lactose operon
- b-galactosidase production
- brake down of lactose
Catabolic repression (glucoseeffect), 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:
- no regulation
- always present
- Enzymes that are needed during all types of growth
- e.g. those involved in glycolysis
• mRNA:
- Unstable
- half-life ~ 2 min  sub-units
-  new mRNA
• polycistronic mRNA
• monocistronic mRNA
- one operator for several genes
- one operator per gene (in eukaryotes)