the lac operon
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- 1. THE lac OPERON Molecular Biology Lecture No: MB 12 Dr. Aga Syed Sameer CSIR Lecturer (Demonstrator) Department of Biochemistry, Medical College, Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190018. India.
- 2. Regulation of Gene Expression Each cell has almost all the genetic material for its growth and development Some genes will be need to be expressed all the time : are involved in vital biochemical processes such as respiration, metabolism etc Other genes need not to be expressed all the time: are switched on an off at need
- 3. Operons The Operon concept was given by Jacob, Monod and Lwoff in 1961 An operon is a group of genes that are transcribed at the same time (hence, expression also) They usually control an important biochemical process They are only found in prokaryotes
- 4. Operons The operon is actually a unit of gene expression & regulation which consists of: Structural genes: which code for the certain specific enzymes involved in metabolic pathways. Their expression is coordinately controlled by various mechanisms Operator & Promoter (OP) Sequences: which are the control sequences located upstream of the structural genes and which in turn play role in regulating the transcription of the structural genes Regulator gene: which code for the protein that binds to the OP Sequences, to control the structural genes transcription/expression
- 5. The lac Operon The lac operon consists of three functional units: Structural genes: Lac ZYA Operator/Promoter Sequences: Lac O and Lac P Regulator gene: Lac I
- 6. The lac Operon lac Y
- 7. The lac Operon The three structural genes are coded as polycistronic mRNA, which in turn code for three polypeptides One of them is enzyme -galactosidase - which hydrolyses lactose into glucose and galactose Second is Permease - which transports lactose within the cell via its internal channel Third one has unknown function
- 8. Adapting to the environment E. coli can use either Glucose (monosaccharide) or Lactose (disaccharide) for its energy requirements However, Lactose needs to be hydrolysed (digested) first So the bacterium prefers to use Glucose when/wherever it can (from environment)
- 9. Regulation of Lac Operon
- 10. Molecular Aspect Lac I Repressor: Coded by Lac I gene binds to Operator and prevents the Transcription of Lac ZYA. This occurs in complete absence of Lactose /in case of well fed Glucose state.
- 11. Molecular Aspect CAP Activator: It binds to the promoter region of the Lac operon and causes 90o kink in DNA and hence enhances the promoter binding of the RNA Pol. This occurs in complete absence of Glucose as well is case of starved state Lactose as sole source of energy
- 12. Glucose: Low or Absent cAMP High cAMP dimerises CAP CRP/CAP attaches to promoter Expression of Lac ZYA enhances -galactosidase in cell Permease in membrane Lactose will be utilised Lactose: Low or Absent LacI being synthesised LacI Repressor attaches to operator Expression of Lac ZYA is halted -galactosidase in cell Permease in membrane Glucose will be utilised Molecular Aspect
- 13. Four situations are possible 1. When glucose is present and lactose is absent the E. coli does not produce -galactosidase. 2. When glucose is present and lactose is present the E. coli does not produce -galactosidase. 3. When glucose is absent and lactose is absent the E. coli does not produce -galactosidase. 4. When glucose is absent and lactose is present the E. coli does produce -galactosidase Thus -galactosidase is synthesized only in presence of Lactose (As a sole source of carbon)
- 14. 1. When lactose is 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 operonOperator site z y a DNA I O Repressor protein RNA polymeraseBlocked
- 15. 2. When lactose is 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
- 16. 2. When lactose is 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
- 17. 3. When both glucose 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.
- 18. When glucose and 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
- 19. 4. When glucose is absent and lactose is present Another protein is needed, an catabolite activator protein. This stabilises RNA polymerase. The catabolite activator protein only works when glucose is absent. As cAMP is available only in absence of glucose. 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
- 20. 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
- 21. Questions?