1. Regulation of Gene expression by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

2. 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

3. 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

4. Biosynthetic reactions, cont. Feedback inhibition: - The biosynthesis of tryptophan occurs in several steps: Chorismate + glutamine  antranilic acid  B  C  D  tryptophan E5E4E3E2E1 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

5. 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

6. Biosynthetic reactions, cont. Mechanism: POattE1E3E2E5E4 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

7. 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

8. Biosynthetic reactions, cont.

12. Catabolic reactions Catabolic systems are inducible Model system – lactose operon in E. coli The inducer is the available carbon/energy source RPOlacAlacYlacZ 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

13. 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

14. Catabolic reactions, cont.

16. 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

17. 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

18. 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

19. Catabolic repression (glucose- effect), cont. Conclusions: - Katabolite repression – a very useful function in bacteria - forces the bacteria to use the best energy source first

20. 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)