Prokaryotic genetics-48 Flashcards
The Iac Operon
What is the Iac Operon?
A way of regulating genes.
What is an operon?
A group of genes under control of the same promoter. This means genes can be regulated together.
Operons are common in prokaryotes- because of differences in eukaryotes and prokaryotes, only works in prokaryotes.
Polycistronic mRNA encodes more than one protein.
What is an example of operon rRNA?
E. coli has 3 rRNA: 16S, 23S and 5S.
Precursor RNA is transcribed from single promoter.
RNA is cleaved to make individual components.
Not all genes code for protein
How many genes does E. coli have?
4277 genes.
What are housekeeping genes?
Genes that are required to be active all of the time.
E.g. genes involved in replication and transcription.
Why are some genes not constitutively expressed?
Making RNA and proteins is energy costly.
Genes are switched on and off- more energy efficient.
What is proof that not all genes are constitutively expressed?
Bacteria grown in minimal medium- must make all nutrients from inorganic components (except glucose).
Then switch to rich medium-genes for making amino acids ect. now available in medium are switched off.
Is Iac operon constitutively transcribed?
No- it is not constitutively transcribed.
Eat glucose first then other carbon sources, only have machinery for other carbon sources if needed.
What is an example of diauxic growth?
Measuring the number of E. coli cells vs time. Also measuring glucose and lactose conc.
-Glucose is used up before lactose.
-1st growth phase: glucose being used up.
-2nd growth phase: lactose being used up.
-Lag phase: glucose has run out, E. coli cannot grow, turns on its lac genes and starts growing again.
-Therefore, carbon sources are used consecutively not simultaneously.
What is diauxic growth?
There are two growth phases.
What proteins do the Iac genes encode?
IacY- encodes beta-galactoside-permease.
IacZ- encodes beta-galactosidase.
IacA- encodes galactoside acetyl-transferase. Transfers an acetyl group to galactosides and glucosides.
Is the Iac operon normally on or off?
It is normally off.
When the operator (Op) is bound by repressor, the operon is turned off.
What is an inducer? What is the inducer?
Inducer- molecule that turns gene on, it disables the repressor.
Allolactose.
Where does allolactose come from?
It cleaves lactose and can turn it to allolactose.
If there is no LacZ (beta-galactosidase), allolactose cannot be made. No lactose, no allolactose.
How can allolactose formation be catalysed by LacZ if operon is repressed?
Even in repressed state there is a small amount of transcription.
What happens when the glucose is used up and lactose is present?
When allolactose is bound by repressor, the operon is turned on. Binds better to allactose than operon.
RNA polymerase binds the promoter, and make LacZ, LacY, LacA and mRNA.
Why is there a lag phase in diauxic growth?
In theory the genes should be substituted on at the same time, then lactase and glucose are used up at the same time.
In the lag phase genes are transcribed and translated to proteins which are folded.
Effect of induction can be measured after a couple of minutes.
Why are genes not induced at the start?
Glucose suppresses activation.
What is CAP? What does CAP do?
CAP- Catabolite Activator Protein
-RNA polymerase interacts favourably with CAP.
-When allolactose is bound by repressor, the operon is turned on.
-CAP protein interacts with cAMP and binds to DNA.
-Transcription is enhanced ~50x
How does glucose inhibit cAMP production?
cAMP is made by adenylate cyclase.
This enzyme is inhibited by glucose.
When we have glucose in the system, no cAMP, have to wait for glucose to be used up and protein can bind.
When allolactose is bound by repressor, the operon is turned on. But no CAP protein means transcription is slow.
What is catabolite repression?
Glucose inhibits adenylate cyclase.
-No cAMP
-Lac operon is transcribed slowly
Same works for other sugars
Lag phase:
-Allolactate: de-repression, cAMP is made adn transcription is enhanced.
Can this promoter (Iac operon) be used to control other genes?
Can produce protein for biotech e.g. insulin.
Can produce protein to study them e.g. structure and function.
Genetic code is universal
Is the Iac promoter a good choice?
Want a strong promoter to make lots of RNA, but don’t want gene constitutively expressed due to energy cost.
But, growing on lactose is inconvenient. Glucose is more convenient.
Problem: if glucose is there, promoter won’t grow. Lactose is inconvenient, gives it a different compound.
What is the induction of expression?
IPTG is a lactose analogue.
Lactose- would be eaten, so concentration decreases.
IPTG- cannot be metabolised, so concentration stays the same.
What happened to variants of Iac promoter which are used today?
They are insensitive to glucose, so they grow on glucose.
Growth slows down on induction- spends a lot of energy making the recombinant protein.
Adding IPTG increases no. of E. coli cells.
