Lecture 4 Flashcards
(21 cards)
Is the regulatory molecule a repressor or an activator?
Repressor. Non functional mutants will produce beta galactosidase even when lactose is absent. -constitutive mutants
What is the regulatory protein and is the product of what gene?
It is a repressor and is a product of the i (lac) gene.
The repressor protein is encoded by gene i.
It prevents transcription by binding to the operator.
RNA polymerase cannot bind to the promoter -transcription is blocked.
How does the repressor repress?
It binds to a DNA sequence in the operon called the operator.
If the operator is mutated what happens?
The repressor cannot bind to the operator. The lac operon will be transcribed all the time.
What causes the repressor to stop repressing?
A signal molecule signals the presence of lactose.
The signal molecule binds to the repressor and causes it to dissociate from the operator.
If the signal molecule cannot bind to the repressor, when would the lac operon be transcribed?
Never (non-inducible)
What would a double mutant be represented by?
Is
Oc
What happens in an is oc mutant?
The lac operon would be transcribed the whole time. In an oc mutant, the repressor cannot bind to the mutant operator, so has no effect.
What is the signal molecule ?
Allolactose
Outline of the regulatory mechanism
When lactose is absent, repressor protein binds to the operator, blocking transcription.
When lactose is present, allolactose binds to the repressor and causes it to dissociate from the operator, allowing transcription.
What do DNase protection experiments using RNA polymerase and Lac repressor show about the binding sites?
The binding sites overlap.
When the RNA polymerase and Lac repressor bind, these proteins protect the DNA from DNase.
Sequencing the protected DNA shows where the protein binds.
Repressor protein -how does it bind?
Made up of 4 polypeptides and it binds as a dimer (2 polypeptides bind to one operator)
What are the 4 domains of each lac repressor polypeptide?
N-terminal DNA binding domain
Hinge region
Allolactose binding central binding domain
C-terminal oligomeric assembly domain
How many auxiliary operators are there and what are they?
2 additional operators.
O2: 410 bp downstream in lacZ gene
O3: 83 bp upstream.
The original, essential operator is O1
How much does binding of lac repressor to O1 reduce transcription by?
20x
How much does binding of lac repressor to O1 and O2 or O3 reduce transcription by?
1000x
Lac repressor binding is…
Co-operative
The DNA between probably forms a loop
Repressor bound to O1 and O2 what is in a loop?
LacZ gene
Repressor bound to O1 and O3 what is in a loop?
Promoter
What is the function of the hinge region on the repressor?
Holds the DNA binding domains in the correct orientation to bind the operator co-operartively.
What causes the repressor to dissociate in the presence of lactose?
Lactose present -allolactose binds to lac repressor and causes a conformational change (binds to the allolactose binding sites)
Hinge region is flexible and is disordered, the subunits no longer bind co-operatively to the operator.
Binding domains fall off -protein is no longer attached to DNA -the DNA is available for RNA polymerase to transcribe.
