Lecture 4

Question 1

How is the Lac operon regulated in E.coli?

2

Answer 1

• Glucose uptake system inhibits Lac permease (LacY)

- High levels of Glucose leads to low levels of cyclic AMP, CAP/CRIP and turns off the lac operon.

Question 2

What is CAP/CRP:

4

Answer 2

• Catabolite activator protein or cAMP, the cyclicAMP receptor protein.
• CAP is a dimer that binds to cAMP.
• it bends the DNA by interacting directly with RNAP to activate transcription
• It can activate many promoters from different relative positions (even overlapping the promoter)

Question 3

What phenotype do both cya- and cap- mutants show?

2

Answer 3

• Lac- phenotype

- Unable to utilise lactose or galactose, arabinose, maltose and other secondary carbon sources

Question 4

What does the CYA gene code for?

Answer 4

adenylate cyclase, it converts ATP to cAMP

Question 5

IS CAP/CRP a positive or negative regulatory mechanism?

2

Answer 5

• it is a positive regulatory protein because knockout of CAP results in essential basal levels of expression
• it is inducible by cAMP (the small MW effector molecule)

Question 6

What is the genetic identification of the CAP binding site?

2

Answer 6

• two promoter classes found within the promoter region, belonging to lac- mutants
• Class1 (eg L8) and class2 (L157)

Question 7

What do class1 mutations of CAP effect

2

Answer 7

• The CAP binding site (L8)

- They are not independent of the CAP regulatory mechanism

Question 8

What do class2 mutations of CAP effect?

2

Answer 8

• They are in the promoter (L157), so the promoter stops working. It still requires the CAP protein.
• They are independent of the CAP regulatory mechanism.

Question 9

What does CAP bind?

Answer 9

The CAP site on DNA

Question 10

What makes up the RNAP?

Answer 10

Beta, beta prime, two alpha, sigma subunits

Question 11

Sigma subunit of RNAP:

Answer 11

• Promoter recognition

Question 12

Alpha subunit of RNAP:

4

Answer 12

• Two domains
• One within the core of the RNAP
• The other domain waves around on the end of the RNAP
• Interacts with the CAP site.

Question 13

What biochemical evidence is there for CAP-RNAP interaction?

4

Answer 13

• CAP and RANP can interact in solution
• Mutually cooperative interaction in binding to DNA, binding of one enhances binding of the other
• Cross-linking studies show close proximity
• X-ray crytallography

Question 14

What genetic evidence is there for CAP-RNAP interaction?

2

Answer 14

• Cap ‘activation-negative’ mutants, can bind DNA but can’t activate transcription.
• RNAP alpha subunit mutations only affect CAP-dependent promoters (so they can’t activate the Lac operon), with no effect on CAP-independent promoters.

Question 15

CAP and LacI act independently at the lac promoter, how do glucose and lactose respond to cAMP levels?
(grid)

Answer 15

Glucose Lactose
cAMP low + +
cAMP low + -
cAMP high - +

Question 16

Other mechanisms of positive regulation of transcription include:
(1)

Answer 16

Arabinose (Ara) operon

Question 17

What is the arabinose (Ara) operon?

4

Answer 17

• araC- (a positive regulator) mutants are unable to utilise arabinose
• cap- mutants are also unable to utilise arabinose (second positive regulatory mechanism)
• AraC dimer binds to the DNA in bot + and - arabinose
• AraC binds regardless of whether arabinose is bound or not bound.

Question 18

Why is the position of DNA on a DNA strand important (araC context)
(3)

Answer 18

• 5 fold increase in expression when 5 and 15 bases were added, but when adding 10 and 20 bases there was no effect
• This is because the distance is not important, but it must be on the correct side of the DNA.
• It must be on the correct side in order to interact

Question 19

What is the NtrC regulation mechanism?

2

Answer 19

• NtrC is involved in the regulation of nitrogen metabolism as a positive regulator of glnA (glutamine synthetase).
• It is activated by phosphorlation, there is no small MW effect molecule.

Question 20

What s the MerR resistance mechanism?

3

Answer 20

• MerT allows the cell to transport poisonous mercury ions out of the cell.
• MerR binds between -10 and -35 in the absence of Mercury this stabilises the inactive promoter.
• MerR is a positive regulator of MerT. Mercury ions bind to MerR and activate induced expression of this gene by twisting the DNA.

Question 21

LacI

3

Answer 21

• negative
• inducible by allolactose
• reversibly binds DNA inhibiting RNAP intiaiton

Question 22

CAP

3

Answer 22

• positive
• inducible by cAMP
• Reversibly binds DNA and interacts with RNAP

Question 23

TrpR

3

Answer 23

• negative
• repressible by tryptophan
• reversibly binds DNA and inhibits RNAP initiation

Question 24

AraC

3

Answer 24

• positive
• Inducible with arabinose
• alternate binding configurations change DNA loop formation

Question 25

NtrC | 3

Answer 25

- positive - phosphorylation - reversibly binds DNA and inters with RNAP. ATPase activates RNAP

Question 26

MerR | 3

Answer 26

- positive - inducible by mercury ions - alters the configuration of DNA at the promoter, allowing RNAP to bind.

Question 27

What is a helix-turn-helix? | 4

Answer 27

- a common structural motif found within many DNA-binding proteins in prokaryotes - made up of a dimer of eg) TrpR an alpha helix, a turn, and another alpha helix. - the recognition helix sits in the major groove of the DNA, and interacts with base pairs. - The second helix contacts the DNA backbone.

Question 28

What do lacl-d mutations define? | 1

Answer 28

- lacl-d mutations define the helix-turn-helix DNA binding domain - Mutations identify repressor domains - Lac repressor has several domains