Lac operon Flashcards
What are the two types of genes that are expressed?
What is an operon?
What does each gene have?
What are the benefits of polycistronic mRNA?
What is the small molecule involved in regulating the operon?
Housekeeping & for protein-production when nutrients available
Collection of genes controlled by 1 promoter (produces polycistronic mRNA)
Start codon AUG - but all controlled by 1 promoter upstream
Less space in genome, efficient expression, still obtain discrete proteins
Substrate or molecule closely related to 1st enzyme in pathway
In order of position on the operon: what is the
promotor (i)
lac i
promotor
operon
lac z
lac y
lac a
transcription initiator of lac i
repressor protein
transcription initiator
transcription controller
beta galactosidase (breaks down lactose into galactose & glucose)
lactose permease (transport lactose)
thiogalactoside transacetylase (removes toxic metabolites which transported by lac y)
What occurs when lactose is present?
Not present?
Why is this being regulated?
How can you describe the lac operon?
Lac i expresses repressor protein which binds to lactose - RNA polymerase can therefore bind to promotor & express lactose to break it down
Lactose does not bind to repressor- repressor binds to operon & prevents RNA pol binding - no transcription
High concentration of lactose means needs to be broken down - lack of would be wasteful to break it down
inducible
What does the lac operator look like?
What operator binding sites are there?
What does the operator form when both sites are bound?
What is required with the binding sites?
What kind of regulation is it once RNA polymerase has been repressed by the operator?
Near perfect inverted repeat - two repressor molecules can bind either side (so 2 repressors for 1 operator due to half sites)
Primary operator binding site & auxillary
Tetramer - 2 bind at primary & 2 at auxillary (half sites at each)
At least one auxillary & one primary site to be bound for repression
Negative regulation - operon is off
Anomalies of the lac operon:
What is the actual inducer of the operon?
How is it formed?
Transportation of the inducer requires permease (from lac y) - so how can lac y be expressed to transport lactose to make the inducer if the operon is not expressed (no inducer present)?
Allolactose
Lactose broken down by beta galactosidase into allolactose
Low level of expression due to repressor falling off without presence of allolactose - allowing for lac y & z to be expressed
What happens to the lac operon when glucose is present in the cell?
What happens when [glucose] is low? 3 steps
Why is this necessary?
What happens when [glucose] is high?
More favourable inducer so inhibits transcription of lac operon until glucose fully used - so can then use lactose for energy
- IIAglc protein is phosphorylated
- Phosphorylated IIAglc forms complex with adenylyl cyclase for its activation
- Active adenylyl cyclase converts ATP -> cAMP
cAMP helps increase the affinity of RNA polymerase to lac operon promotor to use lactose as an energy source
- Glucose is phosphorylated (not IIAglc)
- So adenylyl cyclase is not activated for ATP -> cAMP
How does cAMP increase the affinity of RNA polymerase for the promotor on the lac operon?
How does the CAP-cAMP complex increase the affinity?
Therefore, what happens when [glucose] is low, [lactose] is high and [cAMP] is high?
What happens if [glucose] is high, [lactose] is low and [cAMP] is low?
What happens if [glucose] is high, [lactose] is high and [cAMP] is low?
Binds to CAP binding site to form cAMP-CAP complex on DNA - interacts with C-terminal domains of alpha subunits of RNAP - alpha subunits which bind to promoter (omega recognises)
CAP bends the DNA sequence - so RNA polymerase binds more efficiently to promoter
cAMP produced, forms CAP-cAMP complex on DNA & interacts with c-terminus RNAP. If [glucose] low, [lactose] is high & lactose binds to repressor, allowing RNAP to transcribe operon
Repressor binds
Fewer CAP-cAMP complexes, so RNAP transcribe in some cases - small amount of lac mRNA
What was the purpose of the cis-trans test of lac operon in merodiploid cells? (merodiploid = one wild type & one mutant chromosome)
What happened when the lac i gene was mutated?
What was the conclusion?
What happened when the operator gene was mutated?
What was the conclusion?
To determine how the repressor & operator acts & how information is passed between chromosomes in the same cell
Lac i gene in wild type produced repressor, but repressor still acted on mutant chromosome - so no lac products produced
Repressor acts in trans (information passed from wild type to mutant)
Wild-type: repressor binds to operator so no lac products
Mutant: repressor couldn’t bind to operator so lac products produced
Operator acts in cis - as the mutation is constitutive
What is the negative regulation of the lac operon?
What is the positive regulation of the lac operon?
Repressor binds to lactose & binds to operator
High lactose & low glucose - phosphorylation of IIAglc & activation AC - cAMP-CAP complex increasing affinity rna pol
