Regulation Of Gene Expression : Prokaryotic Regulation

Front 1

What is the definition of the E.coli lac operon?

Back 1

A group of co-ordinately regulated genes involved in lactose metabolism

Front 2

The E.coli lac operon is a group of co-ordinately regulated genes involved in lactose metabolism. What do these genes make?

Back 2

Enzymes that metabolise lactose

Front 3

What does the lacZ gene encode?

Back 3

Encodes B-galactosidase

Front 4

What reaction does B-galactosidase catalyse?

Back 4

N.B cleaves bond via a hydrolysis reaction

Front 5

What does the lacY gene encode?

Back 5

Lactose permease

Front 6

The lacY gene encodes lactose permease. What does this enzyme do?

Back 6

Gets lactose inside the E.coli cell as lactose is normally outside the cell

Front 7

What does the lacA gene encode?

Back 7

Transacetylase

Front 8

What might the function of transacetylase be? ( this is the enzyme that is encoded by the lacA gene)

Back 8

There to detoxify lactose related molecules by adding an acetyl group to them

Front 9

If E.coli is growing in glucose, will the lac operon genes be expressed? Why?

Back 9

No

It would be a waste of energy to make the enzyme

Front 10

If E.coli is growing in lactose with no glucose, will the lac operon genes be expressed?

Back 10

Yes

Front 11

What is the inducer of the lac operon?

Back 11

(allo)lactose

N.B this is a derivative of lactose. When lactose is transported into the cell, some of it is converted to (allo)lactose

Front 12

In experiments, what is used as a synthetic inducer of the lac operon?

Back 12

IPTG

Front 13

Is IPTG hydrolysed by beta-galactosidase?

Why is this important?

Back 13

No

IPTG is an inducer but not a substrate, therefore as it is not broken down, induction of the lac operon will continue

Front 14

What is X-gal used as?

Back 14

A synthetic substrate (gives blue colour when hydrolysed by B-galactosidase)

Front 15

When X-gal is used as synthetic substrate, what can be deduced if a blue colour is present?

Back 15

B-galactosidase must be active and present

Front 16

What is MUG used for?

Back 16

Used as a synthetic substate (gives a fluorescent product when hydrolysed by B-gal)

Front 17

What is polycistronic mRNA?

Back 17

One mRNA molecule can encode more than one protein

Front 18

What does the lacI gene encode?

Back 18

Encodes lac repressor protein

( this is upstream of the lac operon)

Front 19

The lacI gene is constitutively expressed. What does this mean?

Back 19

It is expressed all the time and is not dependent on particular inducers

Front 20

What does the arrow show?

Back 20

The direction RNA polymerase is moving along and therefore where the 1st nucleotide is copied to make mRNA

Front 21

What does the O on this DNA represent?

Back 21

Operator sequence-recognised and bound by lac repressor

Front 22

What does the P on this DNA represent?

Back 22

Promoter sequence:

bound by RNA polymerase but when lac repressor binds operator RNA polymerase cannot transcribe lac operon genes

Front 23

What does lactose bind to?

Back 23

The lac repressor protein

Front 24

What happens when lactose binds to the lac repressor?

Back 24

The conformation of the lac repressor protein is changed to sit cannot bind the operator

RNA polymerase can transcribe the lac operon genes

(N.B one mRNA encodes all the proteins)

Front 25

What happens when there is a mutation in the lacI gene?

(lac I-)

Back 25

No functional lac repressor

Lac operon constitutively transcribed-even with no lactose inducer

Front 26

What happens when there is a lacO mutation?

Back 26

Operator sequence mutated

Lac repressor protein normal, but does not bind mutated operator sequence

Lac operon constitutively transcribed, even without inducer

Front 27

What happens when there is this kind of mutation?

Back 27

'Super' lac repressor: cannot be bound by lactose

Repressor binds operator constitutively

Lac operon transcription repressed, even in the presence of the inducer

Front 28

What does glucose starvation of E.coli lead to?

Back 28

cAMP synthesis

Front 29

Glucose starvation of E.coli leads to cAMP synthesis. What does the cAMP bind to?

Back 29

Catabolite activator protein ( CAP ) and stimulates it to bind to the lac promoter

Front 30

What does DNA bound CAP interact with?

Back 30

RNA polymerase- increases its affinity for the promoter

Front 31

What happens if glucose is present in an E.coli cell and lactose isn't?

Back 31

Low cAMP levels

CAP protein cannot bind

RNA polymerase not good at binding to promoter

no mRNA

Front 32

What can "footprints" show?

Back 32

Show where proteins bind to DNA in the lac operon transcription control region

Front 33

What happens is glucose is present in a cell and lactose is present in an E.coli?

Back 33

Front 34

What happens if there is no glucose present and lactose is present in an E.coli?

Back 34

Front 35

Back 35

Front 36

In practice, why does "no expression" actually mean an extremely low "basal level" of expression of the lac operon gene?

Back 36

The lactose inducer needs to be able to get into the cell.

(Put E.coli into a medium containing lactose-you need lactose to induce expression of lac operon genes. These include lactose permeate which gets lactose inside the cell. If lac operon genes aren't being expressed and lactose is outside the cell, how did lactose get in to induce expression of lac operon genes. Therefore there is always a little bit of permeate made even when lactose isn't inside the cell.)

Front 37

What is arabinose?

Back 37

A pentose sugar from plant cell walls

Front 38

Where and when is arabinose released?

Back 38

Released in the intestine after plant material is eaten

Front 39

Is arabinose absorbed by the intestine?

Back 39

No

Front 40

Arabinose is not absorbed by the intestine. What is a consequence of this?

Back 40

It is a carbon source for E.coli if glucose is absent

Front 41

What is the ara operon?

Back 41

An inducible operon that contains the genes needed for metabolism of arabinose

Front 42

In the arabinose (ara) operon, a single regulatory protein, araC, carries out both ________ and __________ transcriptional regulation

Back 42

positive

negative

Front 43

araC is equivalent of the ___ _________ protein but works in a slightly different way

Back 43

lac repressor

Front 44

What happens when there is no arabinose present?

Back 44

araC protein monomers bind to araI and araO2

the monomers then link to one another, inducing formation of the DNA loop

(N.B araC protein can bind to several places in the operon system. When there is no arabinose present, arak protein can also bind to itself, forming a loop)

Front 45

What does this loop stop?

Back 45

Stops RNA polymerase (and cAMP-CAP) from accessing the promoter, preventing expression of the operon

Front 46

What happens when arabinose is present?

Back 46

Binds to the araC protein

Breaks the connection between araC proteins at aryl and araO2

This opens up the DNA loop

Front 47

One araC-arabinose complex opens up the DNA loop

What can another araC complex do?

Back 47

Binds to aral and interacts with RNA polymerase, increasing its affinity for P ara (arabinose promoter)

Front 48

When glucose is absent, what does CAP-cAMP bind to?

Back 48

Binds to the CAP binding site

(This helps RNA polymerase to bind efficiently to the promoter and initiate transcription)

Front 49

What does araC-arabinose complex also regulate?

Back 49

Its own expression

Front 50

'Tight’ regulation and large increase in expression on additionof arabinose (up to 300 x basal level in 3 seconds) makes thearaC araBAD system suitable for what?

Back 50

E. coli expression vectors

Front 51

Arabinose operon is very efficiently shut down and very efficiently opened up when arabinose is present in nutrient medium. True or false.

Back 51

True.