1. Enzyme Regulation

2. Constitutive enzymes –Enzymes needed at the same level all of the time Regulated enzymes –Enzymes needed under some conditions but not others –Lac Operon –Enzymes are made to break down lactose only if lactose is present

3. Lac operon Glucose = better carbon and energy source than lactose If glucose is available, Lactose pathway will be inhibited But, if glucose is not available, lactose will be used

4. Lac vs. Trp operon The Lac operon is an inducible operon An enzyme is made only when its substrate is present Typical with catabolic enzymes Example: β galactosidase used to metabolize lactose Trp operon is a repressible operon If the product is present, the enzymes that synthesize this product will not be made

5. Terms Inducer – a substance that initiates enzyme induction; lactose Corepressor – a substance that represses enzyme synthesis; arginine; tryptophan Both are called effectors

6. Why are operons important? Why do we want to control expression of genes? saves energy saves raw materials can respond to changes in the environment

7. Parts of the Lac Operon RNA polymerase –The enzyme which makes the RNA (transcription) Promoter: –A region on the DNA where the RNA polymerase can attach and initiate transcription Operator: –A region on the DNA next to the promoter where the repressor binds –The repressor binds to the operator when lactose is absent When you do not want to break down lactose –The repressor cannot bind to the operator when lactose is present, when RNA polymerase binds to the promoter When you want to break down lactose

8. What happens when the Lac operon is off?

9. What happens when the Lac operon is on?

10. Regulation of Lac operon Binding of repressor to the operator – operon off Also, has positive regulation, binding of cAMP/CAP – upstream from the promoter

11. Lac operon Glucose/Lactose media –When lactose is present it binds to the repressor and induces transcription –But only if CAP protein also binds to the DNA (Catabolic activator protein) http://highered.mcgraw- hill.com/olc/dl/120080/bio27.swf

12. Catabolic Repression Mechanism When glucose runs out, cAMP is produced –cAMP—effector cAMP binds to CAP and CAP binds to DNA –CAP (regulator) activates the transcription of the lac genes When glucose is available, catabolic repression will prevent organism from producing lactose catabolism genes –cAMP synthesis is inhibited by presence of glucose –Even if repressor does not bind to the operator

13. Lac operon Lactose must also bind to the repressor

14. Summary: Regulation of the lac operon

15. Trp operon

16. Tryptophan operon Regulated by two control mechanisms Repressor binding to the operator Attenuation: premature termination of transcription

17. Trp repressor binds to the operator http://highered.mcgraw- hill.com/classware/ala.do?isbn=00724646 31&alaid=ala_661272http://highered.mcgraw- hill.com/classware/ala.do?isbn=00724646 31&alaid=ala_661272

18. Regulation by repressor binding to operator Repressor is only active if tryptophan binds

19. Trp repressor When tryptophan levels are low, repressor does not bind to operator and tryptophan can be made When tryptophan levels are high, tryptophan binds to the repressor and activates it and it binds to the operator (trp = corepressor)

20. Trp repressor

21. In prokaryotes, transcription and translation are physically coupled Premature termination of transcription results in incomplete mRNA –Synthesis of leader sequence = premature termination Tryptophan operon regulated by repressor protein (negative control) and also by attenuation Attenuation occurs due to coupling of transcription and translation

22. Trp Operon and attenuation –When Trp plentiful  leader translated  early termination of transcription –When Trp lacking  leader not fully translated  ribosome stalls waiting for TRP tRNAs and then transcription of trp operon continues

23. Attenuation: Tryptophan Operon Inverted repeat in mRNA of trp operon –Stem loop forms –2 conformations: 3,4 stem loop and 2,3 stem loop –3,4 stem loop = transcription pause and reach termination signal –2,3 stem loop = do not have a termination signal

24. Trp operon Fast translation of domain 1 Domain 2 blocked by ribosome Pairing of regions 3-4 Attenuation of transcription Slow translation of domain 1 Waiting for TRP tRNAs Pairing of regions 2-3 Normal gene transcription

25. Attenuation If Trp is available, RNA is made with 2 TRp codons following region 1; a termination sequence is then reached If Trp is low, RNA is made with 2 Trp codons, synthesis must wait until Trp tRNA is available; region 2 is made with no termination signal

26. Regulation of Trp genes by attenuation http://www.csam.montclair.edu/%7Esmalle y/TrpAttenuation.movhttp://www.csam.montclair.edu/%7Esmalle y/TrpAttenuation.mov

27. When are trp genes produced? Excess Trp –Leader sequence transcribed and translated fully –Leader polypeptide attached to ribosome –Causes 3,4 stem loop conformation, which signals transcription pause site and transcription terminated Trp concentration low –Leader sequence transcribed and translated until trp-rich region is reached  a pause occurs –Pause causes 2,3 stem loop conformation, which is not a termination signal and transcription of trp genes continues

28. Regulators/Effectors Trp operon Trp = corepressor + attenuation Lactose = inducer Lactose prevents repressor binding to operator; Enzymes are made +CAP