Lecture 4 Flashcards Preview

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Flashcards in Lecture 4 Deck (28):
1

How is the Lac operon regulated in E.coli?
(2)

- 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.

2

What is CAP/CRP:
(4)

- 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)

3

What phenotype do both cya- and cap- mutants show?
(2)

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

4

What does the CYA gene code for?

adenylate cyclase, it converts ATP to cAMP

5

IS CAP/CRP a positive or negative regulatory mechanism?
(2)

- 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)

6

What is the genetic identification of the CAP binding site?
(2)

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

7

What do class1 mutations of CAP effect
(2)

- The CAP binding site (L8)
- They are not independent of the CAP regulatory mechanism

8

What do class2 mutations of CAP effect?
(2)

- 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.

9

What does CAP bind?

The CAP site on DNA

10

What makes up the RNAP?

Beta, beta prime, two alpha, sigma subunits

11

Sigma subunit of RNAP:

- Promoter recognition

12

Alpha subunit of RNAP:
(4)

- 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.

13

What biochemical evidence is there for CAP-RNAP interaction?
(4)

- 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

14

What genetic evidence is there for CAP-RNAP interaction?
(2)

- 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.

15

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

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

16

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

Arabinose (Ara) operon

17

What is the arabinose (Ara) operon?
(4)

- 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.

18

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

- 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

19

What is the NtrC regulation mechanism?
(2)

- 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.

20

What s the MerR resistance mechanism?
(3)

- 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.

21

LacI
(3)

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

22

CAP
(3)

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

23

TrpR
(3)

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

24

AraC
(3)

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

25

NtrC
(3)

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

26

MerR
(3)

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

27

What is a helix-turn-helix?
(4)

- 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.

28

What do lacl-d mutations define?
(1)

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