Lecture 3 Flashcards
What is found in the tryptophan biosynthetic operon?
5 genes expressed by the promoter (P) found in the tryptophan operon (O) trpE trpD trpC trpB trpA
trpR- mutants:
- They have constitutive expression of the trp operon, it is expressed all the time regardless of any regulatory influence.
Is TrpR a positive or negative regulatory protein?
- A negative regulator. The operon is expressed when levels of tryptophan are low OR repressed when levels of tryptophan are high
Is TrpR an inducible or repressible mechanism?
- It is a repressible mechanism, because high levels of trp bring about low levels of expression
TrpR binds to the Trp Operator, near the promoter of the operon. What feature does this promoter have?
It has dyad symmetry (5’-> 3’ is symmetrically the same as 3’->5’ with one exception), implying that the active regulatory protein binding the operator is a dimer.
How many promoters does TrpR regulate?
It negatively regulates 3 promoters:
- AroG
- Trp
- TrpR (its self!)
What do the promoters that TrpR regulates have in common?
- They each have a region of dyad symmetry
- They all overlap BUT…
- The position relative to the -10 and the -35 sequences differs.
What is the lac operon?
- A promoter and operator with three structural genes: lacZ, lacY and lacA with the lacI gene.
- A tetramer
What does lacZ do?
It cleaves the genes into their consituents (beta-galactosidase).
What does lacY do?
It is involved in the transport of lactose into the cell (permease).
What does lacA do?
We don’t know (transacetylase)!
What does lacI do?
It synthesises repressor monomers that form tetramers which bind to the operator to block transcription of the operon.
What is the lac repressor made up of?
A dimer of:
- DNA-binding domains
- Core domains 1 and 2
- Oligomerisation domains for interactions between the monomers.
When lactose (a carbon source) is present:
The cell expresses the operon in order to utilise lactose. Lactose, the inducer, binds to the repressor complex so that it can no longer bind to the operator by pushing the DNA binding domains appart. RNApol binds at the promter site and mRNA is transcribed, then translated into the 3 proteins.
What happens when you have a lacI- repressor mutant?
- A non-functional monomer is produced, so even in tetramer form it is defective.
- lacI- gene synthesises a defective repressor that does not bind to the DNA.
- The operon is constitutively expressed.
- Recessive to lacI+
- This is a negative regulatory mechanism (determined by the small MW effector molecule).
What is a lacIs mutant?
- Non-inducible (super-respressed)
- Dominant over lacI+
- The repressor is present, and binding to the operator and it stays there regardless as to whether inducer is added or not
- Cannot bind the inducer
What is a lacI-d mutant?
- Constitutive
- Dominant negative over lacI+ because the mutant monomers interfere with the function of the wild type monomers. One ‘bad’ subunit poisons the tetramer which cannot bind DNA, so the operon is expressed.
- They effect DNA binding.
What function of a Lac monomer is affected in a lacI-d mutant?
A monomer has three binding functions:
- binding inducer - fine because not lacIs
- binding other monomers - fine because monomers interfere with other monomers
- binding DNA - presumably missing
Operator constitutive mutant:
- Repressor cannot bind to the mutant operator
- Dominant over wild type and act in cis (if you add a WT copy of the operon the mutant is still expressed constitutively)
- It only effects expression of genes adjacent to it
Where does RNA polymerase sit on the lac operon?
- Over the +1 to beyond -35 region of DNA, the same area covered by the repressor
The lac operon has multiple operators. What are they?
01: essential for repression
02: in the coding sequence of the lacZ gene
03: further upstream
How do you get full repression of the lac operon?
- binding of operator 01 and 02 or 03.
- if 03 or 02 are mutated there is a 2 - 4X increase in basal expression in the absence of inducer
if 03 AND 02 are mutated there is a 100X increase in basal expression in the absence of inducer - a mutation in 01 massive increase in basal expression
What happens when the repressor binds at the lac repressor?
- It increases RNAP binding to the lac promoter while preventing transcription initiation,
- Allowing RNAP to be bound at the same time as LacI repressor allowing transcription to being immediately upon induction of the operon.
