(l3,4,5) The Lac Operon and other operons

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When must a gene be expressed?
When it is needed and at the correct level
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What is the function of the Lac operon?
To allow E.coli to grow on lactose
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What is lactose?
A disaccharide formed from one galactose and one glucose molecule (galactose and glucose are both isomers)
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What 2 enzymes does E.coli require in order to metabolise lactose?
Lactose permease (transports lactose to the cell) and B-galactosidase (hydrolyses lactose to glucose and galactose)
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What 3 genes are in the lac operon?
lacZ (B-Galactosidase) lacY (Lactose permease) and lacA (B-galactoside transactylase- function unknown)
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How does E.coli use glucose?
Preferentially (more favourably)
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When is lac operon transcribed?
Only when its gene products are needed
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What happens when lactose is not present OR glucose is present?
Lac operon not transcribed, glucose used as metabolite
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What happens when lactose is present?
Hardly transcribed as glucose will be used over lactose
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When would the lac operon be transcribed more?
When glucose is absent and lactose is
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What is the main thing to remember with regulation of the lac operon?
Regulation by glucose is a separate process to regulation by lactose
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Where does most of the evidence for the lac operon come from?
Analysing mutants
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How is the wild type denoted?
+
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How is the mutant denoted?
-
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Give an example of how a mutant and wild type are written
Z+ produces active B-galactosidase, Z- does not
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Wha is common way to test for mutants?
Screen for B-Galactosidase (on agar, BG can convert the colourless artificial substrate X-gal to a blue product, thus bacterial colonies containing active BG are blue)
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What type of regulatory molecule regulates transcription?
Repressor molecule (decreases transcription when lactose is absent)
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How does the repressor molecule repress transcription?
It binds to a specific DNA sequence in the operon which prevents the sequence from being transcribed
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What causes the repressor to stop repressing?
There is a signal molecule that signals the presence of lactose by binding to the repressor
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What will happen to the lac operon if there is a mutation in the repressor?
The lac operon will always be transcribed (constitutive) because of the non-functional repressor protein, it will produce B-galactosidase even when lactose is absent
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Where is the gene for the repressor protein?
Gene mapping showed that the gene for the repressor protein was not on but near the lac operon, this is called the lacI gene (I= inducibility)
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The repressor protein binds to a sequence in the operon, what is the sequence called?
The operator
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What would mutations in the operon cause?
They would be constitutive mutants as it would stop the repressor protein form binding and so the operon would be constantly transcribed
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Where are the constitutive mutants of the operon found?
It is not in the lac operon but directly adjacent (as it is where the repressor protein is bound) however the mutants are different from repressor protein mutants
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What are the mutants of the operator called?
Oc mutants
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What would mutants of the signal molecule cause?
This would be a non-inducible mutant as it would not be able to bind to the repressor molecule so the lac operon would never be transcribed
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What are these mutants called?
Is mutants
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What happens in a Is Oc mutant?
The mutant could be constitutive because the repressor protein can't bind to the operon, so a mutation on the signal mutant would have no effect
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What is the signal molecule?
Allolactose (isomer of lactose), produced from lactose in small amounts by small amounts of B-galactosidase present before induction
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What protects DNA from DNase?
If a protein is bound to a section of DNA, that section of DNA is protected from the degradation by DNase
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What did DNase protection experiments show?
That, using RNA polymerase and the Lac repressor, the binding sites overlap
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How does the repressor protein over come this overlap?
The operator has a symmetrical sequence opposite, two lac repressor polypeptides bind to this and block passage of RNA polymerase
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How many regions does the lac repressor have?
4 discrete domains
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What are the 4 domains?
N-terminal DNA binding domain, Hinge region, Allolactose binging central domain, C-terminal oligomeric assembly domain
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What makes up a repressor protein?
4 subunits - only 2 subunits bind to 1 operator
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What are auxiliary operators?
Along with the original operator there are 2 additional operators (auxiliary operators)
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O1 is the original operator, where is O2?
O2 (410bp) downstream in lacZ gene
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Where is O3?
83bp upstream
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What does the binding of the repressor protein to O1 cause?
Reduces transcription
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What does the binding of the lac repressor to O1 2 and 3 do?
It reduces transcription 1000X
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What type of binding is this?
Cooperative binding and the DNA probably forms a loop
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Repressor bound to O1 and O2
LacZ gene is partially in loop
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Repressor bound to O1 and O3
Promoter is in loop
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Describe what is happening with the lac operon when there is no present?
Lac repressor binds tightly to operators because of cooperative binding between subunits, hinge region holds DNA binding domains in correct orientation to bind operator cooperatively
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What causes the repressor to dissociate in the presence of lactose?
Allolactose binds to lac repressor and causes a conformational change, hinge region is disordered and subunits do not bind cooperatively to the operator
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What is a cis?
A mutant that only affects expression of a gene or operon that is on the same DNA molecule, the regulatory sequence must be attached to target gene or operon
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What is a trans?
A mutant that can affect the expression of a gene or operon in a different DNA molecule , the regulatory sequence does not have to be attached to the target gene or operon
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Is the operator cis or trans?
Cis
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Is the repressor cis or trans?
Trans, the repressor can control an operon in a different DNA molecule, it is a diffusible molecule, consistent with it being a protein
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How is the lac operon controlled via glucose?
Via cyclic AMP, glucose controls cAMP concentration
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Glucose present
cAMP concentrations low
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Glucose not present
cAMP concentrations are high and the enzyme adenylate cyclase is activated
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How many operons does cAMP regulate?
Many, >100 promoters
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Which protein does regulation occur via?
Catabolite activator protein (CAP), which is a dimer
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What happens to the catabolite activator protein in the absence of cAMP?
It is inactive
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What happens in the presence of cAMP?
cAMP binds to CAP and cAMP-CAP activates transcription
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Why is activation needed?
Because the lac promoter is a weak promoter, partially because the sequences at -10 to -35 are non-standard
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Describe the mechanism by which transcription of the lac operon is activated by CAP
CAP-cAMP complex binds to DNA next to the -35 region of promoter, bends DNA 90 degrees, RNA polymerase alpha subunit binds to CAP, this increases binding of RNA polymerase to the promoter so it actives transcription
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General features of lac repressor
Mechanism: repression, when a signal molecule binds, it dissociates from DNA, its specific to the lac operon
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General features of CAP
Mechanism: Activation, when a signal molecule binds, it binds to DNA, affects many operons
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Name some examples of other operons
Catabolic operons, biosynthetic operons
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Catabolic operons
Contains genes for breakdown of metabolites, catabolic operons are turned on when metabolite is present and typically when glucose is absent
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Biosynthetic operons
Contains genes for enzymes of biosynthetic pathways, these are turned on when the supply of product is low
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Name a biosynthetic operon
trp operon, for the amino acid tryptophan
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Describe the mechanism of the trp operon
When tryptophan is present, it binds to the tryptophan repressor and causes it to bind to the trp operator, when its absent, the repressor dissociates from the operator, allowing for transcription
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What bacteria does the bobtail squid contain?
Symbiotic bioluminescence bacteria
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Describe the symbiotic relationship between this bacteria and the bobtail squid
Bacteria are bioluminescent so they disguise the squids shadow, squid offers a good growth environment to the bacteria
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When does the bioluminescent occur?
When the bacteria are at a high density in the light organ of the squid
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How is density measured?
Quorum sensing under the control of the lux operon
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What are the signals of cell density?
Acyl homoserine lactones (AHL)
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Describe the process of quorum sensing with the lux operon
LuxI protein catalyses synthesis of AHL, which diffuses out of the cell, when enough bacteria produce AHL, conc outside cell is high, bacteria also takes up AHL, conc inside and outside rises, AHL binds to LuxR protein, activates bioluminescence gene
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How does LuxR affect the lux operon?
With AHL bound to it, LuxR binds to the Lux box in DNA next to the lux operon promoter and increases binding of RNA polymerase
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What mechanism is this?
Positive feedback- LuxI gene itself is in the lux operon, activation of LuxI transcription increases AHL synthesis
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Give some examples of other quorum sensing
Free-living bioluminescent bacteria (cause 'milky seas'), pathogenic bacteria, biofilm formation
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Describe how pathogenic bacteria use quorum sensing =
To produce digestive enzymes only when the density of bacteria is high
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Which genes do the activator proteins activate?
Genes for cell wall degrading enzymes (plant pathogens), proteases (animal pathogens), enzymes for antibiotic synthesis (kill competitors)
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Other cards in this set

Card 2

Front

What is the function of the Lac operon?

Back

To allow E.coli to grow on lactose

Card 3

Front

What is lactose?

Back

Preview of the front of card 3

Card 4

Front

What 2 enzymes does E.coli require in order to metabolise lactose?

Back

Preview of the front of card 4

Card 5

Front

What 3 genes are in the lac operon?

Back

Preview of the front of card 5
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