10.1 - E.coli Lac Operon Flashcards
When does regulation of biologicial processes occur?
When any process is modulated in its frequency, rate or extent through a chain reaction that allows the control of the process being regulated
What do genes expressed by the Lac operon do?
They will incorporate Lactose (disaccharide) and split it into glucose and galactose that can be metabolically used by the cell.
What are the 3 genes involved in the Lac operon?
LacZ, LacY, and LacA
What does LacZ encode?
Beta -galactosidase.
What is beta-galactosidase?
This is an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose
What does LacY encode?
Beta-galactoside permease
What is beta-galactoside permease?
This is a transmembrane symporter that pumps beta –galactosides including lactose into the cell using a proton gradient in the same direction -> symporter
What does LacA encode?
Beta-galactoside acetyltransferase.
What is beta-galactoside acetyltransferase?
This is an enzyme that transfers an acetyl group from acetyl-CoA to beta –galactosides including lactose; it basically acetylates galactose because having too much galactose poisons the cells by deregulating their metabolism. This enzyme therefore enables a transient detoxification
How many operator binding sites are there?
3
What does the lac operon also have?
A promoter sequence, an operator and a terminator
What is upstream the promoter of the lac operon?
A gene that encodes a Lac repressor (Lacl).
What happens when lactose is not present in terms of LacI activity?
LacI binds to the operator sequence lacO and inhibits the expression of the Lac operon by looping DNA together.
What does the looping of DNA together ensure?
That the bacterium only invests energy in the production of machinery necessary for the uptake and utilization of lactose when lactose is present.
What happens when lactose is present?
Allolactose will bind the repressor leading to an allosteric change in its shape.
What is allolactose?
A disaccharide very similar to lactose.
What is allolactose occasionally produced by?
Beta-–galactosidase (LacZ)
What does galactosidase often do?
Converts lactose to allolactose by causing a change in the bonding pattern between galactose and glucose (transferring 1,4-linkage to form 1,6-linkage -> allolactose formed). This enables allolactose to bind to the repressor, removing the inhibition and thereby enabling the expression of the genes required for lactose breakdown.
What happens if there is a mutation in the first gene (i.e. lacZ that encodes beta-galactosidase)?
The whole system shuts down because the enzyme that is responsible for generating the inducer that will allow activation of the operon is no longer produced.
What happens as a result of allolactose binding to the repressor?
The Lac repressor loses its affinity to the operator sequence. The lac repressor will dissociate from the operator allowing the operon to get expressed
What is needed to express the lac operon
Allolactose
However, allolactose is produced by a gene in the lac operon itself (beta –galactosidase). This means that if there is no beta –galactosidase in the cell to begin with, allolactose is not produced and thus the lac operon cannot be expressed naturally.
What is IPTG?
A lactose analogue that is artificially used to induce Lac expression. It is termed gratuitous inducer because it cannot be metabolized by the cell. IPTG will bind the Lac repressor and allows the expression of the Lac operon.
Where is lactose found and when must E. coli have lactose utilization?
E. coli doesn’t encounter lactose very often in its life cycle
Name an experiment related to the lac operon?
E. coli are growing on a M9 minimal media containing Glucose and Lactose.
What happens at first in the experiment?
E. coli are growing exponentially by using glucose as a nutrient source, lactose is not used. Under normal conditions, cells prefer to use glucose as a carbon source
What happens after the first phase in the experiment?
At some point, glucose will run out from the media and the cells will stop growing. (beta galactosidase made in this period)
What happens after a while in the experiment?
They resume growth by starting to utilize lactose after having turned on lac operon. However, they do not grow as rapidly.
What is present during the first phase of the experiment?
There is lactose in the media so the lac repressor has bound to allolactose and has dissociated from the lac operon, which will allow lactose to be broken down into galactose and glucose that can then be used by the cell. However, the proteins used to utilize lactose are still not synthesized. This is because a 2nd mRNA level of lac operon regulation
What is the 2nd mRNA level of lac operon regulation?
cAMP receptor protein (CRP)
What is cAMP?
A 2nd messenger produced by adenylate cyclase that regulates several operons
How doe cAMP levels vary with glucose?
When glucose levels are high cAMP is low and when glucose levels are low cAMP is high
What happens when cAMP levels rise?
cAMP binds to its receptor (CRP). This leads to a conformational change in CRP that enables it to bind to its target on the lac operon. It binds to the class I promoter region, which is near responsive operons.
What does the flexible subunit of the RNA polymerase do?
It stretches and interacts with CRP. This interaction is fundamental for the initiation of transcription of the lac operon.
What happens with presence of lactose in media?
The repressor is removed from the operator
What happens when glucose levels are low?
High cAMP which leads to interaction between CRP & RNA pol which leads to lac operon being transcribed
What is needed for the expression of the lac operon?
Presence of lactose in media and low glucose levels
What kind of promoter does the lac operon have?
A weak promoter due to weak binding constant (Kb)
What does intrinsic promoter strength encompass?
The strength of binding (affinity of RNA pol to bind to the promoter), as well as the ease with which the pol can undergo conf change to open the DNA and maintain it in unwound state
What occurs in the lac operon as a result of its weak binding constant?
Although the constant for opening up the DNA is relatively strong, the pol is a weak binder so it only remains bound for a short amount of time.
How can you increase the binding of pol to the DNA?
It needs to be given another surface of contact.
How does CRP (RNA pol regulator) influence intrinsic promoter strength?
By acting as an anchor that will increase the strength of binding of RNA pol allowing it to initiate transcription. The binding between CRP and RNA pol is not a very powerful binding (short and small contact) but it makes a huge difference in the context of transcription initiation -> greatly increases the intrinsic promoter strength.
Why does pGX-3T use Ptac promoter and not the original lacZYA operon promoter?
Because Ptac is artificial and is not actiated by cAMP. It is only a lac repressor system
Can lambda phase repressor CI also activate transcription?
If it binds to certain sites then in this position it is like a CRP protein and presents surface to RNA polymerase at Prm promoter which is just right to make polymerase stay longer and activate transcription
What happnes to AraC without arabinose?
Without arabinose, the AraC dimer binds at aral, and araO2 to form a loop. AraC does not interact with RNA polymerase, so araBAD is not transcribed.
What happens to AraC with arabinose?
With arabinose, the AraC dimer binds to aral, and araI2, and interacts with RNA polymerase to permit araBAD transcription
