Negative gene regulation

MICROBIOLOGY

1. NEGATIVE GENE REGULATION – e . coli lac OPERON PRESENTED BY : MOUSAMI JARIA ST. GEORGE COLLEGE OF MANAGEMENT AND SCIENCE MSC MICROBIOLOGY SEMESTER 2

2. WHAT IS GENE EXPRESSION? It is the process by which information from a gene is used in the synthesis of a functional gene product. When the information stored in our DNA is converted into instructions for making proteins or other molecules it is called gene expression .

3. TYPES OF GENE EXPRESSION There are mainly two types of gene regulation/ expression : A. Positive regulation B. Negative regulation.

4. POSITIVE REGULATION When the expression of genetic information is quantitatively increased by the presence of specific regulatory element , it is called as positive regulation . The element or molecule mediating positive regulation is called positive regulator. Eg : E . coli ara operon

5. NEGATIVE REGULATION When the expression of genetic information is quantitatively decreased by the presence of specific regulatory element, it is called as negative regulation . The element or molecule mediating positive regulation is called negative regulator. Eg : E . coli lac operon .

6. LACTOSE (L ac ) operon Lac operon is an operon or a group of genes with a single promoter ( transcribed as single mRNA). The genes in the operon encode proteins that allow bacteria to use lactose an energy source. It is an inducible catabolic operon of E . coli. It consists of: 1. Structural genes: It carries 3 structural genes Z, Y & A. Codes respectively for : beta- galactosidse , galactoside permease & thiogalactoside transacetylase .

7. lac operon model

8. WHAT MAKES lac OPERON TURN ON? E . coli can break down lactose , but if glucose is available they use that. Glucose requires fewer steps and less energy to break down than lactose. Howevr if lactose is the only sugar available, E . coli uses it as energy source To use lactose, the bacteria must express the lac operon genes, which encode key enzymes for lactose uptake and metabolism . Only when 2 conditions are met: Lactose is available. Glucose is not available

9. Two regulator proteins are involved: The lac repressor, acts as lac sensor Catabolite activator protein (CAP), acts as a glucose sensor These proteins bind to the DNA of the lac operon and regulate its transcription based on lactose and glucose levels .

10. STRUCTURE The lac operon contains 3 genes : lacZ , lacY , & lacA . These genes are transcribed as single mRNA, under control of one promoter . lacZ encodes an enzyme called beta- galactosidase which is responsible for splitting lactose into readily usable glucose and galactose . lacY gene encodes a membrane protein called lactose permease , which is a transmembrane ‘’pump’’ that allows the cell to import lactose.

11. The lacA gene encodes an enzyme known as transacetylase that attaches a particular chemical group to target molecules lac operon also contains a number of regulatory DNA sequences. These are regions of DNA to which particular regulatory proteins can bind , controlling transcription of operon . The promoter is the binding site for RNA polymerase , the enzyme that performs transcription .

12. The operator is a negative regulatory site bound by the lac repressor protein . The operator overlaps with the promoter and when the lac repressor is bound , RN polymerase cannot bind to the promoter and start transcription . The cap binding site is a positive regulatory site that is bound by catabolite activator protein(CAP). When CAP is bound to this site , it promotes transcription by helping RNA polymerase bind to the promoter .

13. THE lac repressor The lac repressor is a protein that represses (inhibits) transcription of the lac operon . It does this by binding to the operator, which partially overlaps with the promoter. When bound the lac repressor gets in RNA polymerases way and keeps it from trancribing the operon . .

14. WHERE DOES THE lac REPRESSOR COME FROM ? When lactose is not available , the lac repressor binds tightly to the operator , preventing transcription by RNA polymerase But when lactose is present , the lac repressor lose its ability to bind DNA. It floats off the operator , clearing the way for RNA polymerase to transcribe the operon . This change in the lac repressor is caused by the small molecule allolactose ,an isomer of lactose

15. When lactose is available , some molecules will be converted to allolactose inside the cell. Allolactose binds to lac repressor and makes it change shape so it can no longer bind DNA. Allolactose is an example of an inducer , a small molecule that triggers expression of a gene operon . The lac operon is considered an inducible operon because it is usually turned off(repressed) , but can be turned on in the presence of inducer allolactose .

16. CATABOLITE ACTIVATOR PROTEIN(CAP) When lactose is present , the lac repressor loses its DNA binding ability . This clears the way for RNA polymerase to bind to the promoter and transcribe the lac operon RNA polymerase alone does not bind very well to the lac operon promoter. It might makes a few transcripts , but it won’t do much more unless it gets help from catabolite activator protein(CAP) CAP binds to region of DNA just before the lac operon promoter and helps RNA polymerase attach to promoter, driving high levels of transcription.

17. WHERE DOES CAP COMES FROM? CAP is not always active (able to bind DNA), instead its regulated by a small molecule cyclic AMP( cAMP ). cAMP is a ‘’ hunger signal’’ made by E. coli when glucose levels are low. cAMP binds to CAP , changing its shape and making it able to bind DNA and promote transcription. Without cAMP , CAP cannot bind DNA and is inactive.

18. HOW IS c AMP MADE , AND HOW DOES IT REPORT GLUCOSE LEVELS? cAMP is only active when glucose levels are low ( cAMP levels are high). Thus the lac operon can only be transcribed at high levels when glucose is absent. This strategy ensures that bacteria only turn on the lac operon and start using lactose after they have used up all of the preferred energy source (glucose).

19. summary GLUCOSE PRESENT LACTOSE ABSENT : No transcription of lac operon occurs because lac repressor remains bound to the operator and prevents transcription by RNA polymerase Also cAMP levels are low because glucose levels are high , so CAP is inactive and cannot bind DNA

20. 2. GLUCOSE PRESENT LACTOSE PRESENT : Low level transcription of the lac operon occurs The lac repressor is released from the operator because the inducer ( allolactose ) is present. cAMP levels are low because glucose is present CAP remains inactive and cannot bind to DNA , so transcription only occurs at a low leaky level.

21. 3. GLUCOSE ABSENT , LACTOSE ABSENT : No transcription of lac operon occurs. cAMP levels are high because glucose levels are low , so CAP is active and will bound to DNA Lac repressor will also be bound to the operator acting as a roadblock to RNA polymerase and preventing transcription .

22. 4. GLUCOSE ABSENT, LACTOSE PRESENT : Strong transcription of the lac operon occurs The lac repressor is released from the operator because the inducer ( allolactose ) is present cAMP levels are high because glucose is absent ,so CAP is active and bound to the DNA . CAP helps RNA polymerase bind to the promoter , permitting high levels of transcription.

23. summary

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