10.1 - E.coli Lac Operon

Question 1

When does regulation of biologicial processes occur?

Answer 1

When any process is modulated in its frequency, rate or extent through a chain reaction that allows the control of the process being regulated

Question 2

What do genes expressed by the Lac operon do?

Answer 2

They will incorporate Lactose (disaccharide) and split it into glucose and galactose that can be metabolically used by the cell.

Question 3

What are the 3 genes involved in the Lac operon?

Answer 3

LacZ, LacY, and LacA

Question 4

What does LacZ encode?

Answer 4

Beta -galactosidase.

Question 5

What is beta-galactosidase?

Answer 5

This is an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose

Question 6

What does LacY encode?

Answer 6

Beta-galactoside permease

Question 7

What is beta-galactoside permease?

Answer 7

This is a transmembrane symporter that pumps beta –galactosides including lactose into the cell using a proton gradient in the same direction -> symporter

Question 8

What does LacA encode?

Answer 8

Beta-galactoside acetyltransferase.

Question 9

What is beta-galactoside acetyltransferase?

Answer 9

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

Question 10

How many operator binding sites are there?

Answer 10

3

Question 11

What does the lac operon also have?

Answer 11

A promoter sequence, an operator and a terminator

Question 12

What is upstream the promoter of the lac operon?

Answer 12

A gene that encodes a Lac repressor (Lacl).

Question 13

What happens when lactose is not present in terms of LacI activity?

Answer 13

LacI binds to the operator sequence lacO and inhibits the expression of the Lac operon by looping DNA together.

Question 14

What does the looping of DNA together ensure?

Answer 14

That the bacterium only invests energy in the production of machinery necessary for the uptake and utilization of lactose when lactose is present.

Question 15

What happens when lactose is present?

Answer 15

Allolactose will bind the repressor leading to an allosteric change in its shape.

Question 16

What is allolactose?

Answer 16

A disaccharide very similar to lactose.

Question 17

What is allolactose occasionally produced by?

Answer 17

Beta-–galactosidase (LacZ)

Question 18

What does galactosidase often do?

Answer 18

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.

Question 19

What happens if there is a mutation in the first gene (i.e. lacZ that encodes beta-galactosidase)?

Answer 19

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.

Question 20

What happens as a result of allolactose binding to the repressor?

Answer 20

The Lac repressor loses its affinity to the operator sequence. The lac repressor will dissociate from the operator allowing the operon to get expressed

Question 21

What is needed to express the lac operon

Answer 21

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.

Question 22

What is IPTG?

Answer 22

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.

Question 23

Where is lactose found and when must E. coli have lactose utilization?

Answer 23

E. coli doesn’t encounter lactose very often in its life cycle

Question 24

Name an experiment related to the lac operon?

Answer 24

E. coli are growing on a M9 minimal media containing Glucose and Lactose.

Question 25

What happens at first in the experiment?

Answer 25

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

Question 26

What happens after the first phase in the experiment?

Answer 26

At some point, glucose will run out from the media and the cells will stop growing. (beta galactosidase made in this period)

Question 27

What happens after a while in the experiment?

Answer 27

They resume growth by starting to utilize lactose after having turned on lac operon. However, they do not grow as rapidly.

Question 28

What is present during the first phase of the experiment?

Answer 28

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

Question 29

What is the 2nd mRNA level of lac operon regulation?

Answer 29

cAMP receptor protein (CRP)

Question 30

What is cAMP?

Answer 30

A 2nd messenger produced by adenylate cyclase that regulates several operons

Question 31

How doe cAMP levels vary with glucose?

Answer 31

When glucose levels are high cAMP is low and when glucose levels are low cAMP is high

Question 32

What happens when cAMP levels rise?

Answer 32

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.

Question 33

What does the flexible subunit of the RNA polymerase do?

Answer 33

It stretches and interacts with CRP. This interaction is fundamental for the initiation of transcription of the lac operon.

Question 34

What happens with presence of lactose in media?

Answer 34

The repressor is removed from the operator

Question 35

What happens when glucose levels are low?

Answer 35

High cAMP which leads to interaction between CRP & RNA pol which leads to lac operon being transcribed

Question 36

What is needed for the expression of the lac operon?

Answer 36

Presence of lactose in media and low glucose levels

Question 37

What kind of promoter does the lac operon have?

Answer 37

A weak promoter due to weak binding constant (Kb)

Question 38

What does intrinsic promoter strength encompass?

Answer 38

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

Question 39

What occurs in the lac operon as a result of its weak binding constant?

Answer 39

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.

Question 40

How can you increase the binding of pol to the DNA?

Answer 40

It needs to be given another surface of contact.

Question 41

How does CRP (RNA pol regulator) influence intrinsic promoter strength?

Answer 41

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.

Question 42

Why does pGX-3T use Ptac promoter and not the original lacZYA operon promoter?

Answer 42

Because Ptac is artificial and is not actiated by cAMP. It is only a lac repressor system

Question 43

Can lambda phase repressor CI also activate transcription?

Answer 43

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

Question 44

What happnes to AraC without arabinose?

Answer 44

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.

Question 45

What happens to AraC with arabinose?

Answer 45

With arabinose, the AraC dimer binds to aral, and araI2, and interacts with RNA polymerase to permit araBAD transcription