3) Operons Flashcards
What happens to the levels of lac operon when glucose is present?
Diauxic growth:
1) High concentration of glucose= growth occurs exponentially
2) But then concentration of glucose decreases= Lactose kicks in and starts to induce the lac operon= Transcription ON
3) Beta-galactosidase activity then increases as more is produced= Growth resumes
Diauxie is used to describe this
Glucose= Prevents the induction of operons concerned with catabolism= No break down
If glucose is present when lactose is present= Lactose operon is not induced
What does it mean by “glucose is the preferred carbon source”?
When E.coli is grown on both glucose and lactose= Glucose is utilised first
ONLY when nearly all of the glucose is used up, is the lac operon induced
What does Adenyl cyclase do?
Also known as adenylate cyclase
Enzyme that catalyses the cyclisation of ATP into cyclic AMP
What effect does glucose have on adenyl cyclase?
GROWTH on glucose (not glucose itself) inhibits the enzyme= low cAMP levels
High glucose= Low cAMP
What does the mutants of adenyl cyclase do?
Cya mutants= Have defects in enzyme= Unable to make cAMP
Unable to induce any of the sugar operons e.g. lac, gal, ara EVEN WHEN GLUCOSE IS ABSENT
Addition of cAMP= immediately restored operon inducibility
RESULTS: cAMP must be needed for induction of the operon
How is the catabolism of glucose able to repress (prevent induction) the lactose operon?
1) Phosphoenolpyruvate: glucose phosphotransferase system protein complex imports and phosphorylates sugars simultaneously. Transports glucose into the cell
2) Protein IIA-Glc (specific for glucose) becomes Dephosphorylated when glucose is present
3) Dephosphorylated version able to inhibit the activity of adenyl cyclase
4) Results in no cAMP being produced as cAMP is added for transcription of the operon= Transcription is SWITCHED OFF= Lac operon does not work
5) IF NO GLUCOSE IS PRESENT= no transport will occur, protein IIA-Glc will stay phosphorylated= stimulates adenyl cyclase= produces cAMP= TRANSCRIPTION ON
What protein does cAMP bind to?
Binds to protein that can also bind to lac DNA
Protein: CRP= cAMP Receptor Protein
Also called CAP (catabolite gene activator protein)
How does cAMP aid transcription?
1) Glucose level is low, cAMP level is high
2) cAMP attaches to CAP
3) CAP can attach to lac gene promoter= facilitates binding of RNA polymerase
4) Transcription ON
CAP is able to help RNA polymerase: Bending of DNA around CAP= unwinding stress that helps with melting of DNA in Pibnow box
NO interaction between DNA pol and CAP protein but CAP protein MUST BE PRESENT
IF both glucose and lactose are avaliable=CAP is detached and lac operon is not transcribed EVEN though lactose is present= Effect of glucose takes precedence over everything
Why is the lac promoter slightly unusual?
Most bacterial promoters have TATAAT in their pribnow box (-10)
BUT lac promoter: TATGTT
Result= Makes a G-C link= Joined by 3 bonds rather than only 2 for A-T= Harder to melt G-C bond
-10 box MUST be melted first to open up the DNA to be transcribed
SUMMARY: What happens when lactose is present for regulation of lac operon?
Presence of lactose= Represses the repressor= Induces lac operon= Transcription ON= Lactose can be broken down
SUMMARY: What happens when glucose is present EVEN when lactose is also present?
Operon is normally OFF:
When glucose is present. Transcription is OFF as no cAMP to bind to CAP protein
IF CAP is absent= operon is NOT switched on= Transcription is OFF
WHEN CAP is present, the operon can still be repressed by the lac repressor= When no lactose is available
What are biosynthetic operons?
How do they work?
Expression of related enzymes that synthesis small molecules
MUCH MORE ENERGY SAVING to use pre-existing essential compounds found in growth media =IF SOMETHING IS ALREADY THERE= Enzymes which encode enzymes to synthesis of the molecules will be SWITCHED OFF
If not included in growth media, the bacterial must synthesis EVERYTHING= Operons are switched ON
What is the structure of the tryptophan operon?
Codes for the protein: Tryptophan Synthetase which MAKES tryptophan
Trp operon: 5 structural genes: trpE, trpD, trp C, trpB and trpA
Operon has promoter and operator + transcribed region before the first structural gene (leader region)
What is the regulatory gene for the tryptophan operon? How does it work?
Gene: trpR
Encodes the top repressor protein
OPPOSITE TO THE LAC OPERON:
Tryptophan is present in growth media= Bacteria do not need to synthesise it= Trp operon is suppressed= TRANSCRIPTION OFF WHEN Tryptophan is present
How does the trp repressor become active?
By itself: Inactive= Aprorepressor
Needs to complex with tryptophan itself (corerepressor) before it is active
1) Trp repressor + tryptophan= Holorepressor (complete)
2) Holorepressor then binds to the trp operator, trpO
3) Inhibits transcription by Steric hindrance= SAME as lac operon
What is the difference between positive and negative control for repressor and activators?
Negative control= repressor
- Induction: Inactivating the repressor
- Repression= Letting the repressor do the work
Positive control= Activator
- Induction= Letting the activator do the work
- Repression= Stopping the activator working
What type of control do lac and trp operons work by?
Work by negative control
IF lacI and trpR genes are deleted or inactivated= Constitutive expression of operons
Absence of a regulatory control, the operons are transcribed and translated therefore to regulate them, we have to switch them OFF
What happens to operons which are regulated by positive control?
REQUIRE SWITCHING ON
If the gene for a regulatory protein is inactivated= Operon is never expressed
How is regulation by positive control can be done in bacterial systems and also eukaryotes?
An activator protein is required to switch ON transcription
By itself, the activator protein will NOT WORK and needs another co-regulatory molecule to bind to it before it can activate transcription
Repressor (unbound) + Operator DNA Repressor (bound)/ Operator DNA
In order to get 100% inhibition (by formation of Repressor (bound)/Operator DNA), the concentration of Repressor (unbound) must also increase by several orders of magnitude= But cannot have such high concentrations of protein for every regulatory protein in a single bacterial cell= Around 99% repression
Example: CAP/cAMP, Ara
In eukaryotes: Postive agent binds the activator and makes it inactive
Why is positive control usually used in multicellular organisms?
Cannot have just 99% inhibition= As they are much more precise controlled developmental pathways in multicellular organisms
Absence of the activator protein= No expression at all
What does the Arabinose Operon do?
Can break down TL-arabinose which is another sugar which can be catabolised if there is NO glucose available as energy source
D-xylose-5 phosphate is an intermediate of the pentose phosphate pathway which can be used to release energy
What is the arabinose operon regulated by?
BOTH negative and positive control + has 2 regulatory DNA sequences: araO and araI
How is the arabinose operon regulated when arabinose is absent?= Negative Control
1) NO arabinose:
AraC protein acts as a repressor
2) AraC binds to the operator araO + araI
3) Causes DNA to form a loop
4) Repression of transcription of the 3 structural genes (araBAD)= Transcription OFF= No catabolism
How is arabinose operon regulated when arabinose is PRESENT?= Positive control
Presence:
1) Arabinose binds to AraC protein and converts it to an activator protein
2) Binds to araI site
3) Switches ON transcription of the 3 genes
Switching on the operon also requires cAMP-CAP
