Lecture 4 Flashcards
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.
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)
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
What does the CYA gene code for?
adenylate cyclase, it converts ATP to cAMP
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)
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)
What do class1 mutations of CAP effect
2
- The CAP binding site (L8)
- They are not independent of the CAP regulatory mechanism
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.
What does CAP bind?
The CAP site on DNA
What makes up the RNAP?
Beta, beta prime, two alpha, sigma subunits
Sigma subunit of RNAP:
- Promoter recognition
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.
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
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.
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 - +
Other mechanisms of positive regulation of transcription include:
(1)
Arabinose (Ara) operon
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.
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
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.
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.
LacI
3
- negative
- inducible by allolactose
- reversibly binds DNA inhibiting RNAP intiaiton
CAP
3
- positive
- inducible by cAMP
- Reversibly binds DNA and interacts with RNAP
TrpR
3
- negative
- repressible by tryptophan
- reversibly binds DNA and inhibits RNAP initiation
AraC
3
- positive
- Inducible with arabinose
- alternate binding configurations change DNA loop formation
NtrC
3
- positive
- phosphorylation
- reversibly binds DNA and inters with RNAP. ATPase activates RNAP
MerR
3
- positive
- inducible by mercury ions
- alters the configuration of DNA at the promoter, allowing RNAP to bind.
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.
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
