Theme 3- Module 2 (Prokaryotic transcriptional regulation)

Question 1

What two proteins increase in amount when E.coli cells transition from glucose to lactose metabolism?

Answer 1

Beta-galactosidase

Lactose permease

Question 2

True or false: beta-galactosidase and lactose permease are detectable in small amounts when glucose is the primary energy source

Answer 2

False

They are not expressed until glucose is fully depleted from the growth medium

Question 3

What is lactose permease?

Answer 3

Transmembrane protein that allows for the transport of lactose into the bacterial cells

Question 4

What is beta-galactosidase?

Answer 4

Cytoplasmically situated bacterial enzyme that cleaves the imported lactose into glucose and galactose

Question 5

What is a key advantage to the organization of the prokaryotic genome?

Answer 5

Groups of related genes with similar functions can often be found clustered together into operons. Leads to the ability to control the transcription of the whole gene cluster as one unit.

Question 6

What do operons consist of?

Answer 6

A promoter

An operator (or on-off switch)

The coordinated gene cluster whose products will function in a common pathway or cellular response

Question 7

What is an operator?

Answer 7

A sequence of nucleotides near the start of the operon that can allow or inhibit transcription

Question 8

How do operators regulate transcription?

Answer 8

When the operator is not bound to any transcriptional inhibitor, then the RNA polymerase can attach to the promoter and transcribe the genes in the operon

Question 9

What does the lac operon control in E.Coli?

Answer 9

The regulation of beta- galactosidase and lactose permease expression

Question 10

What are the regulatory sequences of transcription in the lac operon?

Answer 10

• Promoter that binds the RNA polymerase complex

- Operator (lacO) which is the binding site for a repressor protein

Question 11

What is the name of the sequence that the operator is expressed by?

Answer 11

Lac I coding sequence

Question 12

What are the two main structural genes that code for the primary proteins needed to facilitate lactose metabolism?

Answer 12

lacY

lacZ

Question 13

What does the lacY gene code for?

Answer 13

A lactose permease transport protein

Question 14

What does the lacZ gene code for?

Answer 14

B-galactosidase

Question 15

Which gene controls the expression of the lacZ and Y genes?

Answer 15

lac l

Question 16

What does lac I code for?

Answer 16

A repressor protein which can bind to the operator and inhibit transcription from occurring

Question 17

The ability of a repressor protein to halt transcription in this manner (the way that lac I halts transcription) is often identified as ________

Answer 17

Negative transcriptional regulation

Question 18

How does negatively regulated transcription work?

Answer 18

Repressor protein will bind to the operator region of the operon

RNA polymerase is no longer able to bind to the promoter region of the operon

Results in turning off transcription

Question 19

Is the lactose operon negatively or positively regulated?

Answer 19

Negatively

Question 20

How does negatively regulated transcription work in the lac operon specifically?

Answer 20

Repressor protein, which is encoded by the lac l gene is constitutively expressed at low levels

(Repressor protein binds with the lac O operator region and the RNA polymerase complex is not able to bind to the promoter)

This is the state of the lac operon activity in most cases when E. coli cells are exposed to glucose

Question 21

Describe the structure of the repressor in the lac operon

Answer 21

Tetrameric protein

- four identical protein subunits that binds tightly to operator regions on the lac operon DNA

Question 22

How is the repressor able to prevent RNA Poly from binding?

Answer 22

Once all four subunits bind the lac operon DNA, that the DNA is twisted into a loop and RNA Poly is not able to bind

Question 23

How does the presence of glucose in the growth medium affect the repressor?

Answer 23

Facilitates the constitutive expression of the repressor protein (and thus inhibits expression of lactase proteins)

Question 24

How are repressor proteins allosterically inhibited by lactose?

Answer 24

Lactose = inducer molecule

• binds to the repressor proteins
• causes a change in the conformation of the repressor so that it can no longer bind to the DNA

Question 25

What should you look out for when you’re looking at the general nutritional state of E. coli cells? (i.e. what’s a good indicator)

Answer 25

The conc of cAMP

Question 26

When glucose levels are high in the environment surrounding bacterial cells, there is an inhibition of the enzyme __________

Answer 26

Adenylyl cyclase

Question 27

What does adenylyl cyclase do?

Answer 27

Catalyzes the production of cAMP from ATP

Question 28

When glucose levels are low, what will the conc of cAMP be like? Why?

Answer 28

High bc increased activity of the adenylyl cyclase enzyme

Question 29

Describe the basic steps of positive regulation of the lac operon

Answer 29

1) Low levels of glucose –> high cAMP levels
2) cAMP binds to CRP (causes conformation change0
3) CRP-cAMP complex binds to CRP-cAMP binding site on DNA
3) activate transcription

Question 30

If there was both glucose and lactose in the environment, would the CRP-cAMP complex bind to the lac operon?

Answer 30

No

Question 31

Can transcription occur if the activator is not present or is not able to bind to the activator binding site?

Answer 31

No

Question 32

What do inducer molecs do?

Answer 32

Allosterically inhibit repressor proteins so it can no longer bind to DNA

(bind and cause a conformation change)

Question 33

What is the inducer molec involved in the lac operon?

Answer 33

Lactose (binds to the repressor)