Lac Operon

1. Lac Operon

2. Lactose and Glucose Much of the control of gene expression occurs at the transcriptional level Our understanding of transcriptional regulation comes from studies of enzyme induction in E.coli E. Coli exhibit an extremely sophisticated regulation of enzyme Induction in response to changing environmental conditions. The primary source of food for bacteria is glucose! If both glucose and lactose are present together, glucose is utilized first. The organism will first breakdown glucose by turning on genes for enzymes that metabolize glucose. The enzymes required for lactose metabolism are shut off! Once the glucose is completely metabolized, the genes responsible for glucose metabolism are shut down. Then the genes for the enzymes involved in lactose metabolism are turned on. How does a cell turn on and off these genes? How does E. coli monitor the environment?

3. Gene Regulation Questions were first addressed by studying genes controlling lactose metabolism in E. coli Lactose metabolism requires a b-galactosidase When only lactose is present, the genes for lactose metabolism are turned on. When all the lactose is broken down, these enzyme are shut off That is the genes coding for these enzymes are shut off

4. Gene Regulation Cells– lactose -galactosidase 1 very low level Cells + lactose -galactosidase 1000 induced There are two ways you can visualize this occurring. Lactose present gene ON Lactose absent gene OFF

5. Gene Regulation You can classify genes in a simple way in two classes 1) Structural 2) Regulatory The structural genes are those that produce the enzyme required for lactose metabolism Permease Allows transport of lactose into the cell Galactosidase cleaves lactose into glucose and galactose Transacetylase Unknown function The regulatory loci determine whether transcription of the structural genes will occur. They monitor and respond to Environmental conditions (presence of lactose) The loci that regulate lactose metabolic enzymes include The repressor gene The operator The promoter P O Z Y A Promoter Operator Structural genes

6. LACTOSE (lac) OPERON P Repressor The operator is a specific DNA sequence to which the repressor Binds The promoter is a specific DNA sequence to which the RNA Polymerase binds By binding to the operator, the lac repressor prevents transcription of the structural genes LacZ, LacY and LacA. 3 Structural genes Permease Y galactosidase Z transacetylase A 3 Regulatory elements Operator O promoter P repressor I

7. The Lac operon The structural genes and the regulatory elements form a functional Genetic unit called the Lac Operon. The repressor controls whether a RNA polymerase will transcribe The lac operon genes The repressor protein has a high affinity for binding the operator DNA. If repressor is bound to operator, the structural genes are not Transcribed because the repressor physically blocks RNA polymerase from transcribing the adjacent genes. If repressor is not bound to the operator, the RNA polymerase Can transcribe the structural genes How is the operon regulated with respect to the environment? When lactose is present in the media, the operon is ON and When lactose is absent in the media, the operon is OFF.

8. Repressor Repressor Repressor Repressor The lac repressor The repressor is the key element in regulating the operon with respect to environmental conditions The repressor has two functional sites DNA binding site Lactose binding site When the repressor is bound to lactose, it no longer binds to the operator DNA. Binding of lactose to the repressor alters the conformation of the repressor protein so that it no longer has a high affinity for the operator. Lactose No Lactose The repressor is an example of an allosteric protein. That is a protein that changes from one conformation to another These changes alter the function of the protein.

9. No lactose present

10. Lactose present

12. Bacteria and Genetics Jacob and Monod were the first to propose the operon model of gene regulation following genetic analysis of E. coli Griffiths; Fig 5-2, pg. 153 Genetically testing the lac operon model requires complementation analysis which requires diploids E. Coli are prokaryotes- by definition a haploid

13. Mechanism of DNA transfer Partial diploids can be created in E.coli through the use of F’ Factors. It involves Nonreciprocal (one way) transfer through F pilus encoded by F factor Jacob and Monod generated F’ factors carrying various parts of the Lac Operon These were used in complementation tests between other factors in the operon Griffiths; Fig 5-7, pg. 157

14. Pseudodiploid a- b- c- A+ B+ c- a- b- c-

15. Analysis of lac operon mutants using partial diploids Use of bacterial genetics to demonstrate existence of promoters and repressors Introduce F factors carrying mutations at the Lac operon Induce operon transcription I P A O Y Z

16. I P A O Y Z Repressor Repressor Analysis Genotype galactosidase(Z) Permease (Y) no lac lac no lac lac ---------------------------------------------------- I+ P+O+Z+Y+ - + - + I- P+O+Z+Y+ + + + + Z+ and Y+ are coordinately expressed. Both are induced or not induced together, because both are transcribed on a single mRNA The I repressor is required to prevent Lac gene expression in the absence of lactose. The operon is negatively controlled. Its basal state is ON and It must be actively turned OFF by binding of repressor to operator

17. Repressor I+ Analysis I P A O Y Z Repressor Genotype galactosidase(Z) Permease (Y) no lac lac no lac lac ---------------------------------------------------- I- P+O+ Z+Y+ + + + + I- P+O+ Z+Y+/F(I+) - + - + Experiment with partial diploid demonstrates whether a gene is CIS or TRANS dominant (That is whether the gene product is Diffusible or not) I- P A O Y Z

18. Repressor Analysis I P A O Y Z Repressor Genotype galactosidase(Z) Permease (Y) no lac lac no lac lac ---------------------------------------------------- I+ P+O+Z+Y+/ - + - + F(I+ P+O+Z-Y+) I+ P+O+Z+Y+/ - + - + F(I+ P+O+Z+Y-) I+ P+O+Z-Y+/ - + - + F(I+ P+O+Z+Y-) Complementation of the structural genes still occurs I P A O Y Z

19. I I I P P P A A A O O O Y Y Y Z Z Z mRNA mRNA mRNA Analysis Z- cell Y- cell

20. Is mutant A new repressor mutant was discovered called Is This mutant prevents induction of the lac structural genes Genotype galactosidase(Z) no lac lac ---------------------------------------------------- I+P+O+Z+Y+ - + I- P+O+ Z+Y+ + + Is P+O+ Z+Y+ - - Is P+O+ Z+Y+/F(I+) - - Is P+O+ Z+Y+/F(I-) - - I+ P+O+ Z+Y+/F(Is) - - The Is mutation is dominant to I+ and I- How can this mutant be explained?

21. Is mutant A new repressor mutant was discovered called Is This mutant prevents induction of the lac structural genes Genotype galactosidase(Z) no lac lac ---------------------------------------------------- Is P+O+Z+Y+ - - The Is mutant eliminates the lactose binding site on the repressor The repressor is always bound to the operator and blocks transcription. The presence of lactose in the media does not cause it to fall off the DNA.

22. Is Is Is I+ I+ I+ Is mutant Genotype galactosidase(Z) no lac lac ---------------------------------------------------- I+P+O+Z+Y+/F(Is) - - The Is mutant eliminates the lactose binding site on the repressor The Is mutant is dominant to I+ How do you explain this? I+ P A O Y Z Is There is a 50:50 ratio of Is to I+ in the cell

23. Is Is Is I+ I+ I+ Is mutant I+ P A O Y Z Is There is a 50:50 ratio of Is to I+ in the cell So at random you would expect 50% of the operators to be bound by I+ and 50% by Is If this were the case then you should see half the operons being induced and galactosidase being made (50%) This does not occur The reason is as soon as lactose binds and releases the normal repressor (I+), the Is mutant repressor irreversibly binds the operator.

24. Repressor Repressor LacO The operon model also proposes a direct interaction between the Repressor and a specific DNA site on the DNA (operator) The repressor binds the operator and physically prevents RNA Polymerase from transcribing the structural genes I P A O Y Z

25. Lac Operator mutants Genotype galactosidase(Z) Permease (Y) no lac lac no lac lac ---------------------------------------------------- I+P+O+Z+Y+ - + - + I+P+Oc Z+Y+ + + + + I+P+Oc Z+Y+/F(O+) + + + + I+P+O+Z+Y+/F(Oc) - + - + I-P+O+Z+Y+ + + + + Results of #3 suggests that Oc is dominant to O+ Results of #4 suggests that O+ is dominant to Oc The allele of O (Oc or O+) this is directly adjacent to the structural genes (in the cis configuration) determines the expression pattern of the structural genes. This is called Cis-dominance in contrast to the Trans- dominance pattern observed for the I gene. The Oc mutation only disrupts transcription of genes directly adjacent to it (on the same DNA). It has no effect on the transcription of genes on other chromosomes The repressor (I) is a diffusible protein (Trans dominant). The operator (O) is a DNA sequence that binds the repressor. It is not diffusible. The repressor cannot bind under any circumstances because the operator sequence is altered. Oc is dominant but only in Cis

26. I+ LacO The Oc mutation only affects the expression of those genes Adjacent and on the same chromosome. It has no effect on The expression of genes on other chromosomes I Oc P A O Y Z No Txn I P A Oc Y Z Txn In general genes that exhibit Trans-dominance produce a diffusable protein and regions of the DNA to which proteins binds exhibit a Cis-dominance.

27. Catabolite repression Catabolite Repression of the lac operon- When we introduced the subject of regulation of gene expression, I gave an example of the response of E. coli when placed in a media containing both glucose and lactose. The induction of the lac operon does not occur until all the glucose has been metabolized. E. coli contains a mechanism that favors glucose uptake first. Once the glucose is gone, the lactose will be utilized. This result is not predicted by the lac operon model we presented. According to the model, lactose is solely responsible for the regulation of this operon. Input from other environmental factors, such as the presence of glucose, could not be accounted for. Eventually it was realized that some catalytic breakdown product of glucose prevented the activation of the lac operon even in the presence of lactose. This effect was termed: Catabolite repression.

28. CAP In addition to negative regulation by the lac repressor, the lac operon also required positive regulation. That is, a factor was needed for specific activation of the operon. The specific activator was called CAP ( Catabolic activator protein) produced by the gene crp (not part of the lac operon). CAP forms a complex with cyclic AMP (cAMP). This complex is capable of activating the lac operon. There is an inverse relationship between the amount of glucose in the cell and the amount of cAMP/CAP. High glucose levels result in Low cAMP/CAP levels Low glucose levels result in High cAMP/CAP levels When glucose levels are high, little cAMP will be available to form the CAP-cAMP activating complex of the lac-operon     In the positive control by the cAMP/CAP complex, the lac operon becomes responsive to levels of glucose in the media.

29. CAP mediated activation of the lac operon How does the cAMP/CAP complex regulate the lac operon. This complex binds to a region of the promoter and is required for RNA polymerase binding. The lac operon possesses both negative regulation (inducer/repressor) and a positive control system (cAMP/CAP system).