Download to read offline
![Dual Regulation of lac operon
Negative control by lac repressor >> needs the
inducer (lactose) to inactivate the lac repressor
Positive control by CAP (activated by high [cAMP]
resulting from low [glucose]) >> determines rate of
transcription if the operator is NOT blocked by the
repressor](https://image.slidesharecdn.com/08-lac-operon-3-241129022315-c96b485e/75/08-lac-lactose-operon-molecular-OPERON-3-pptx-31-2048.jpg)
08-lac-lactose operon molecular OPERON-3.pptx
- 1. THE lac OPERON ©2007 Paul Billiet ODWS
- 2. Operons An operonis a group of genes that are transcribed at the same time. They usually control an important biochemical process. They are only found in prokaryotes. © NobelPrize.org Jacob, Monod & Lwoff © 2007 Paul Billiet ODWS
- 3. The control ofgene expression Each cell in the human contains all the genetic material for the growth and development of a human Some of these genes will be need to be expressed all the time These are the genes that are involved in of vital biochemical processes such as respiration Other genes are not expressed all the time They are switched on an off at need © 2007 Paul Billiet ODWS
- 4. Lac Operon TheLac Operon is an example of an operon that is able to regulate itself depending on the environmental conditions it is subjected to. It codes for 3 genes: Beta-galactosidase, lactose permease and Thiogalactosidase transacetylase. These genes are involved in lactose metabolism. If lactose is absent, the system is turned off; if lactose is present, the operon is switched on. The bacteria’s favoured source of food is glucose and if that is present the operon does not need to be switched on, as there is no need to metabolise lactose.
- 7. Element Purpose Operator (LacO)Binding site for repressor Promoter (LacP) Binding site for RNA Polymerase Repressor Gene encoding the lac repressor protein. Binds to DNA at the operator & blocks binding of RNA Pol at the promoter. LacI Controls production of the repressor protein
- 8. Enzyme Function Beta galactosidaseThis enzyme hydrolyzes the bond between the two sugars, glucose and galactose. It is coded for by the gene LacZ. Lactose Permease This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose. It is coded for by the gene LacY Thiogalactosidase transacetylase The function of this enzyme is not known. It is coded for by the gene LacA.
- 11. The operatoris a short region of DNA that lies partially within the promoter and that interacts with a regulatory protein that controls the transcription of the operon. The regulatory gene lacI produces an mRNA that produces a Lac repressor protein, which can bind to the operator of the lac operon. The lac repressor is a dimeric protein that can join to form a tetramer. In the absence of lactose, the repressor tightly b inds to the operator. The presence of the repressor prevents RNA polymerase from unwinding the DNA strand to initiate transcription.
- 12. The lac Operon The lac operon consists of three genes each involved in processing the sugar lactose One of them is the gene for the enzyme β- galactosidase This enzyme hydrolyses lactose into glucose and galactose © 2007 Paul Billiet ODWS
- 13. The metabolism oflactose in E. coli & the lactose operon •To use lactose as an energy source, cells must contain the enzyme b-galactosidase. •Utilization of lactose also requires the enzyme lactose permease to transport lactose into the cell. •Expression of these enzymes is rapidly induced ~1000-fold when cells are grown in lactose compared to glucose. QuickTime™ and a GIF decompressor are needed to see thispicture. IPTG: non- metabolizable artificial inducer (can’t be cleaved) Trans- glycosylation LacZ: -galactosidase; Y: galactoside permease; A: transacetylase (function unknown). P: promoter; O: operator. LacI: repressor; PI and LacI are not part of the operon.
- 14. The Lactose Operon lacZ: b-galactosidase lacY : lactose (galactoside) permease lacA : galactoside transacetylase
- 15. Adapting to theenvironment E. coli can use either glucose, which is a monosaccharide, or lactose, which is a disaccharide However, lactose needs to be hydrolysed (digested) first So the bacterium prefers to use glucose when it can © 2007 Paul Billiet ODWS
- 16. Lac operon Phenomenon: 1. Onglucose- Bacteria grow fine 2. On lactose- bacteria don’t grow, then grow because they induce an enzyme that breaks lactose down into glucose b-galactosidase 3. ON lactose AND glucose- No b-galactosidase !
- 17. How the Lacoperon works…. The Genes and Regulatory elements
- 19. Four situations arepossible 1. When glucose is present and lactose is absent the E. coli does not produce β-galactosidase. 2. When glucose is present and lactose is present the E. coli does not produce β-galactosidase. 3. When glucose is absent and lactose is absent the E. coli does not produce β-galactosidase. 4. When glucose is absent and lactose is present the E. coli does produce β-galactosidase © 2007 Paul Billiet ODWS
- 20. The control ofthe lac operon © 2007 Paul Billiet ODWS
- 21. 1. When lactoseis absent A repressor protein is continuously synthesised. It sits on a sequence of DNA just in front of the lac operon, the Operator site The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing Regulator gene lac operon Operator site z y a DNA I O Repressor protein RNA polymerase Blocke d © 2007 Paul Billiet ODWS
- 22. 2. When lactoseis present A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site z y a DNA I O © 2007 Paul Billiet ODWS
- 23. 2. When lactoseis present A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site Promotor site z y a DNA I O © 2007 Paul Billiet ODWS
- 24. 3. When bothglucose and lactose are present This explains how the lac operon is transcribed only when lactose is present. BUT….. this does not explain why the operon is not transcribed when both glucose and lactose are present. © 2007 Paul Billiet ODWS
- 25. When glucoseand lactose are present RNA polymerase can sit on the promoter site but it is unstable and it keeps falling off Promotor site z y a DNA I O Repressor protein removed RNA polymerase
- 26. 4. When glucoseis absent and lactose is present Another protein is needed, an activator protein. This stabilises RNA polymerase. The activator protein only works when glucose is absent In this way E. coli only makes enzymes to metabolise other sugars in the absence of glucose Promotor site z y a DNA I O Transcription Activator protein steadies the RNA polymerase © 2007 Paul Billiet ODWS
- 27. Summary Carbohydrates Activator protein Repressor protein RNA polymerase lac Operon +GLUCOSE + LACTOSE Not bound to DNA Lifted off operator site Keeps falling off promoter site No transcription + GLUCOSE - LACTOSE Not bound to DNA Bound to operator site Blocked by the repressor No transcription - GLUCOSE - LACTOSE Bound to DNA Bound to operator site Blocked by the repressor No transcription - GLUCOSE + LACTOSE Bound to DNA Lifted off operator site Sits on the promoter site Transcription © 2007 Paul Billiet ODWS
- 28. Catabolite Repression Why doesn’tbeta-galactosidase get induced in medi containing Glucose?
- 29. When cAMP ispresent: When cAMP is absent: RNA polymerase bound tightly to promoter (blue DNA) RNA polymerase bound loosely to promoter (blue DNA) FREQUENT TRANSCRIPTION INFREQUENT TRANSCRIPTION cAMP CAP CAP site CAP CAP site Operator Operator lacZ lacY lacZ lacY lacA lacA cAMP binds to CAP and the cAMP-CAP complex binds to DNA at the CAP site. RNA polymerase binds the promoter efficiently. Transcription occurs frequently. CAP does not bind to DNA. RNA polymerase binds the promoter inefficiently. Transcription occurs rarely. CAP regulates lac operon positively and requires cAMP for DNA binding
- 30. Glucose inhibits theactivity of the enzyme adenylyl cyclase, which catalyzes production of cAMP from ATP. The amount of cAMP and the rate of transcription of the lac operon are inversely related to the concentration of glucose. ATP Adenylyl cyclase Glucose inhibits this enzyme cAMP Two phosphate groups Infrequent transcription of lac operon (Cell continues to use glucose as energy source.) CAP does not bind to DNA CAP LOW cAMP INACTIVE adenylyl cyclase HIGH glucose concentration LOW glucose concentration ACTIVE adenylyl cyclase HIGH cAMP cAMP CAP CAP-cAMP complex binds to DNA Frequent transcription of lac operon (Cell uses lactose if lactose is present.) Cyclic AMP (cAMP) is synthesized when glucose levels are low
- 31. Dual Regulation oflac operon Negative control by lac repressor >> needs the inducer (lactose) to inactivate the lac repressor Positive control by CAP (activated by high [cAMP] resulting from low [glucose]) >> determines rate of transcription if the operator is NOT blocked by the repressor
- 32. Figure 17-15 lac operon PromoterRepressor INFREQUENT TRANSCRIPTION INFREQUENT TRANSCRIPTION CAP site CAP site CAP site FREQUENT TRANSCRIPTION Operator Operator Operator RNA polymerase bound loosely to promoter RNA polymerase bound loosely to promoter RNA polymerase bound tightly to promoter Glucose HIGH Glucose HIGH Glucose LOW Lactose LOW Lactose HIGH Lactose HIGH lacZ lacZ lacZ lacY lacY lacY lacA lacA lacA Inducer-repressor complex
- 33. Catabolite Repression oflac Operon (Positive regulation) • When excess glucose is present, the lac operon is repressed even in the presence of lactose. • In the absence of glucose, the lac operon is induced. • Absence of glucose results in the increase synthesis of cAMP • cAMP binds to cAMP regulatory protein (CRP) (AKA CAP). • When activated by cAMP, CRP binds to lac promoter and stimulates transcription.
- 34. Molecular Cell Biology,4th Edition, Lodish et. al. (2000)
- 36. Why does theLac Operon need an activator? Lac promoter has lousy promoter!!!
- 37. Lac Z LacY Lac A P O Pi I Pcrp crp Regulation of the Lac Operon: Low lactose, High glucose Transcription from Pcrp and Pi is constitutive: always expressed in an unregulated fashion. Active repressor binds to operator and prevents RNA polymerase from reaching structural genes. Active repressor crp mRNA I mRNA Inactive CAP protein No mRNA produced No Z, Y, A proteins produced Pol
- 38. Regulation of theLac Operon: High lactose, High glucose Lac Z Lac Y Lac A P O Pi I Pcrp crp Active repressor crp mRNA I mRNA Inactive CAP protein Lactose Lactose (inducer) binds to the repressor and inactivates it. RNA polymerase transcribes Lac Z, Y and A at low frequency. + Inactive repressor Pol Z, Y, A mRNA Transcription Translation B-galactosidase Permease Acetylase
- 39. Regulation of theLac Operon: High lactose, Low glucose Translation B-galactosidase Permease Acetylase Lac Z Lac Y Lac A P O Pi I Pcrp crp Active repressor crp mRNA I mRNA Inactive CAP protein Lactose + Inactive repressor + cAMP is produced when glucose levels are low. cAMP activates CAP. Active CAP binds to the promoter to increase RNA polymerase binding. RNA polymerase transcribes Lac Z, Y and A at HIGH frequency. cAMP Active CAP protein Pol Z, Y, A mRNA Transcription
- 40. Regulation of theLac Operon: Low lactose, Low glucose Active repressor Lac Z Lac Y Lac A P O Pi I Pcrp crp crp mRNA I mRNA Inactive CAP protein cAMP + Active CAP protein Although RNA polymerase binding is enhanced by Active CAP, the operator is blocked by active repressor. RNA polymerase cannot transcribe Z, Y and A. No mRNA produced No Z, Y, A proteins produced Pol
- 41.
Editor's Notes
- #28 Glucose can block the production of b-galac.
- #29 High levels of glucose---NO CAMP OR CAP! If lactose, the repressor releases and binds to lactose. CAP allows RNA pol. To bind really good to the DNA and then, if glucose is present—no cap. And not good binding to DNA and not very often transcription. cAMP and CAP make transcription more efficient and this can only happen if NO glucose. Makes sense cuz you really need the transcription of lacZ and lacY because then you have Adenyl cyclase is the molecule that lets glucose in and if no glucose, it changes the structure of ATP and dephosphorylates ATP into cyclic-AMP.
- #30 Remember what the cell wants! In the presence of high glucose, you have low cAMP. Which is what you want. If glucose level is low, you get high cAMP. And cyclic AMP is needed for CAP. And CAP pushes the synthesis of the lac operon.