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Genes: Organisation and Function > Lecture 4 > Flashcards

Lecture 4 Flashcards

1
Q

How is the Lac operon regulated in E.coli?

2

A
  • 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.

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2
Q

What is CAP/CRP:

4

A
  • 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)
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3
Q

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

2

A
  • Lac- phenotype

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

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4
Q

What does the CYA gene code for?

A

adenylate cyclase, it converts ATP to cAMP

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5
Q

IS CAP/CRP a positive or negative regulatory mechanism?

2

A
  • 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)
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6
Q

What is the genetic identification of the CAP binding site?

2

A
  • two promoter classes found within the promoter region, belonging to lac- mutants
  • Class1 (eg L8) and class2 (L157)
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7
Q

What do class1 mutations of CAP effect

2

A
  • The CAP binding site (L8)

- They are not independent of the CAP regulatory mechanism

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8
Q

What do class2 mutations of CAP effect?

2

A
  • 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.
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9
Q

What does CAP bind?

A

The CAP site on DNA

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10
Q

What makes up the RNAP?

A

Beta, beta prime, two alpha, sigma subunits

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11
Q

Sigma subunit of RNAP:

A
  • Promoter recognition
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12
Q

Alpha subunit of RNAP:

4

A
  • 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.
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13
Q

What biochemical evidence is there for CAP-RNAP interaction?

4

A
  • 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
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14
Q

What genetic evidence is there for CAP-RNAP interaction?

2

A
  • 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.
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15
Q

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

A

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

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16
Q

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

A

Arabinose (Ara) operon

17
Q

What is the arabinose (Ara) operon?

4

A
  • 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
Q

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

A
  • 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
Q

What is the NtrC regulation mechanism?

2

A
  • 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
Q

What s the MerR resistance mechanism?

3

A
  • 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
Q

LacI

3

A
  • negative
  • inducible by allolactose
  • reversibly binds DNA inhibiting RNAP intiaiton
22
Q

CAP

3

A
  • positive
  • inducible by cAMP
  • Reversibly binds DNA and interacts with RNAP
23
Q

TrpR

3

A
  • negative
  • repressible by tryptophan
  • reversibly binds DNA and inhibits RNAP initiation
24
Q

AraC

3

A
  • positive
  • Inducible with arabinose
  • alternate binding configurations change DNA loop formation
25
Q

NtrC

3

A
  • positive
  • phosphorylation
  • reversibly binds DNA and inters with RNAP. ATPase activates RNAP
26
Q

MerR

3

A
  • positive
  • inducible by mercury ions
  • alters the configuration of DNA at the promoter, allowing RNAP to bind.
27
Q

What is a helix-turn-helix?

4

A
  • 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
Q

What do lacl-d mutations define?

1

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

Genes: Organisation and Function (33 decks)

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