Lecture 5 Flashcards
What types of signals are there?
2
- Small MW molecules (typrophan, cAMP, arabinose)
- Environmental signals (chemicals, light, osmolarity, temperature, nutrition
Describe the two parts of the Two-component sensing mechanism:
(3)
- A transfer of phosphatase between two components to activate gene regulation
- Component 1: at the cell membrane receives the signal (input transmits the signal to the cytoplasm (inside)
- Component 2: receives the signal from the membrane, mediates response via gene regulation, often a DNA binding domain
Component 1:
2
- Input domain outside the membrane in periplasmic space
- Transmitter domain in the cytoplasm, but attached to the membrane.
Transmitter domain of component 1 activities:
2
Two opposing activities:
- Kinase (K): adds a phosphate group to a specific Histidine on itself - transmits signal
- Phosphatase (P): removes a phosphate group from Component 2 - stops signal transmission
Receiver domain of component 2:
- P is transferred to a specific Aspartate on the receiver domain of component 2.
- The output domain of component 2 is activated to regulate gene expression.
The two component sensing mechanism order of P transmission:
input signal -> input domain(C1) autophosphorylates -> cell membrane -> transmitter (C1) transfers the phosphates to C2-> receiver (C2) -> output (C2) -> output domain regulates gene activity
If the input signal is removed, what happens?
Component 1 adopts Phosphatase (P) activity, removes P group from receiver domain of component 2 and the output domain of component 2 is inactivated.
eg 1: response to osmolarity
- Porins (proteins that form pores in the membrane) size influences solute flow into the cell, controlling osmolarity. The pore size is changed by expression of two porin genes, which depend on the osmolarity of the external environment.
- Nitrogen levels in the environment,
Under high osmolarity what will happen?
- the membrane domain transfers the phosphate to the DNA-binding domain, regulating porin gene expression.
Under low osmolarity what will happen?
- The pore size will change. The second component changes back to the phosphatase activity, by actively removing the phosphatase activity on the other receiver. Expression is reversed.
eg 2: response to nitrogen levels
Outer domain sense N levels. If low the cell expressed glutamine synthetase which allows greater N incorporation.
- Low N sensed -> phoyphorylates itself -> K transferred to regulatory protein domain with 3 domains, 1 recieves phosphate group, 1 is DNA binding domain, 1 is ATPase domain which interacts with RNAP -> activation of glnA expression.
- When reversed, glnA gets switched off by taking the phosphatase activity, (P)
NtrC
Regulatory protein that, when activated, binds to cis-acting sites upstream of glnA, allowing activated transcription of the glnA gene.
glutamate + NH4 -glutamine synthetase>
-> glutamine
The major pathway of nitrogen into the cell.
What is glutamine and what are it’s two promoters:
3
- an AA required for protein synthesis.
- Promoter 1 (basal promoter) transcribed with sigma70RNAP
- Promoter 2 (activated when high levels of glnA is needed) transcribed with sigma54RNAP when
NtrC binds to cis-acting sites
What is a transcriptions terminator?
2
In e.coli there are two types:
- Factor independent/intrinsic
- Factor-dependant TF
What is an example of a factor independent (or intrinsic) terminator?
(2)
- An independent terminator is a structure in the mRNA (it has already been transcribed by the mRNA) that signals to the RNAP to pause.
- eg) regions that have dyad symmetry causing stem and loops to form, followed by a string of U residues in RNA, RNAP stops, and the weaker A-U bp assist with transcript release
What is an example of a factor-dependant terminator?
3
- Rho factor-dependant transcription terminator
- A hexameric protein that uses ATP to bind to mRNA. It unwinds the RNA-DNA duplex to unwind.
- It binds at rut (Rho UTilisation) sites at the 5’ end, with high C and low G in mRNA which precedes the termination point.
- Reaches RNAP and causes it to dissociate.
In prokaryotes is transcription and translation coupled?
1
- Yes! no nucleus and cytoplasm so it happens all at the same time
In prokaryotes can premature termination of translation can cause premature termination of transcription to occur?
(1)
- Yes!
eg) - In a WT 100% activity will occur in the lacZYA genes.
- In a nonsense mutation of lacZ, premature termination of lacZ will occur, but lacY and lacA have separate promoters so we would expect normal expression levels.
- BUT they all have premature termination of transcription by RNAP in the downstream genes.
What is polarity?
The action of an upstream mutation in a gene is having an effect on the expression of a downstream gene.
Ribosomes and Rho and termination:
3
- Ribosomes prevent Rho from premature termination, at least until the ribosome reaches it’s stop codon.
- Then the Rho protein can reach the RNAP causing termination.
- When the ribosome pauses it gives the Rho protein time to catch up.
Attenuation of transcription in the Trp operon:
- Trp operon has 5 structural genes (TrpR binds at the operator, and the attenuator is a transcription terminator).
There are two deletion mutations in the Trp operon that both have different effects, what are they?
- DeltaLD102: interferes with the operator, so an increase in expression is observed.
- DeltaED53: effects a different regulatory mechanism
