1. The need for gene regulation Bacterial genome4,000 genes Human genome100,000 genes Not all expressed at any one time May need very high levels e.g. translation elongation factors May need very low levels e.g. some DNA repair enzymes Expression needs to vary with time and cell type - otherwise every cell would be the same and there would be no organisms except microbes

2. TERMINOLOGY Housekeeping genes: required all the time Constitutive expression: gene constantly switched on Inducible gene: gene switched on only in defined conditions Repressible gene: gene switched off in defined conditions

3. E. COLI PROMOTER CONSENSUS SEQUENCE n17 TATAATTTGACA n5-9 (-10 region)(-35 region) Consensus sequence has highest affinity for RNA polymerase - and highest frequency of transcription initiation

4. THE TRYPTOPHAN OPERON EDCBA Operator (binding site for repressor) promoter trp A,B,C,D,E are genes that code for enzymes of tryptophan synthesis

5. Tryptophan absent inactive repressor gene on Tryptophan present active repressor gene off tryptophan promoter/ operator start site promoter/ operator RNA polymerase binds to promoter

6. NEGATIVE REGULATION OF THE TRP OPERON Operator sequence lies within the trp promoter sequence In absence of tryptophan the repressor protein is unable to bind to the operator hence RNA polymerase can bind to the promoter and initiate transcription In presence of tryptophan, 2 molecules of tryptophan bind to the repressor protein allowing it to bind to the operator. This prevents RNA polymerase from binding to the promoter, hence transcription is blocked.

7. Cell Membrane Outside Inside Lactose Galactose + Glucose -galactosidase Allolactose Galactoside permease

8. The lac operon PiIPOZYA Pi : promoter for I gene P : promoter for lac Z, A, Y genes O : operator for lac Z, A, Y genes Z : -galactosidase Y : galactoside permease A : thiogalactoside transacetylase

9. LAC OPERON IN REPRESSED STATE PIPI I PO ZY A DNA mRNA Repressor No transcription

10. lac operon is subject to negative regulation In absence of lactose the lac operon is repressed - only a few copies of -gal present in the cell. In presence of lactose the lac operon is induced. Mutations in I gene (upstream of lac operon) or operator cause constitutive expression of lac operon gene products. If I gene is defective, can introduce correct copy of I gene on another DNA molecule and recover gene repression. i.e. I gene product is a diffusible product that represses the lac operon

11. INDUCTION OF THE LAC OPERON mRNA A. No lactose: repressor bound to operator - inhibits transcription P PO lac genes B. lactose present: release of repressor/ inducer complex - allows transcription O RNA polymerase binds

12. Negative regulation (bound repressor inhibits transcription) A) Molecular signal causes dissociation of regulatory protein from DNA DNA

13. Negative regulation (bound repressor inhibits transcription) B) Molecular signal causes binding of regulatory protein to DNA DNA

14. POSITIVE REGULATION OF LAC OPERON Glucose is preferred carbon source If glucose is present, lac operon is repressed - even if lactose is present This is catabolite repression Repressive effect mediated via cAMP and a protein called catabolite gene activator protein or CAP In absence of glucose CAP binds to site near promoter and enhances transcription 50 fold

15. Low glucose, low lactose High glucose, low lactose (low cAMP) PO lac genes CAP PO lac genes CAP Lac repressor (lacI)cAMP-CAP No transcription

16. Low glucose, high lactose High glucose, high lactose (low cAMP) PO lac genes CAP PO lac genes CAP Transcription No transcription RNA polymerase

17. Promoter consensus sequence TTGACATATAAT -35-10 lac promoter TTTACATATGTT -35-10 lac promoter is weak RNA polymerase binding site

18. Summary of Lecture 2 Negative regulation - active form of repressor protein binds to operator, inhibiting transcription Trp operon (tryptophan biosynthesis) repressor protein binds to trp operator when tryptophan is present Lac operon - repressor protein binds to lac operon when lactose is absent Lac operon also subject to positive regulation - in absence of glucose CAP binds upstream from lac operon greatly enhancing transcription