Lecture 5

Question 1

What types of signals are there?

2

Answer 1

• Small MW molecules (typrophan, cAMP, arabinose)

- Environmental signals (chemicals, light, osmolarity, temperature, nutrition

Question 2

Describe the two parts of the Two-component sensing mechanism:
(3)

Answer 2

• 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

Question 3

Component 1:

2

Answer 3

• Input domain outside the membrane in periplasmic space

- Transmitter domain in the cytoplasm, but attached to the membrane.

Question 4

Transmitter domain of component 1 activities:

2

Answer 4

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

Question 5

Receiver domain of component 2:

Answer 5

• 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.

Question 6

The two component sensing mechanism order of P transmission:

Answer 6

input signal -> input domain(C1) autophosphorylates -> cell membrane -> transmitter (C1) transfers the phosphates to C2-> receiver (C2) -> output (C2) -> output domain regulates gene activity

Question 7

If the input signal is removed, what happens?

Answer 7

Component 1 adopts Phosphatase (P) activity, removes P group from receiver domain of component 2 and the output domain of component 2 is inactivated.

Question 8

eg 1: response to osmolarity

Answer 8

• 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,

Question 9

Under high osmolarity what will happen?

Answer 9

• the membrane domain transfers the phosphate to the DNA-binding domain, regulating porin gene expression.

Question 10

Under low osmolarity what will happen?

Answer 10

• 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.

Question 11

eg 2: response to nitrogen levels

Answer 11

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)

Question 12

NtrC

Answer 12

Regulatory protein that, when activated, binds to cis-acting sites upstream of glnA, allowing activated transcription of the glnA gene.

Question 13

glutamate + NH4 -glutamine synthetase>

Answer 13

-> glutamine

The major pathway of nitrogen into the cell.

Question 14

What is glutamine and what are it’s two promoters:

3

Answer 14

• 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

Question 15

What is a transcriptions terminator?

2

Answer 15

In e.coli there are two types:

• Factor independent/intrinsic
• Factor-dependant TF

Question 16

What is an example of a factor independent (or intrinsic) terminator?
(2)

Answer 16

• 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

Question 17

What is an example of a factor-dependant terminator?

3

Answer 17

• 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.

Question 18

In prokaryotes is transcription and translation coupled?

1

Answer 18

• Yes! no nucleus and cytoplasm so it happens all at the same time

Question 19

In prokaryotes can premature termination of translation can cause premature termination of transcription to occur?
(1)

Answer 19

• 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.

Question 20

What is polarity?

Answer 20

The action of an upstream mutation in a gene is having an effect on the expression of a downstream gene.

Question 21

Ribosomes and Rho and termination:

3

Answer 21

• 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.

Question 22

Attenuation of transcription in the Trp operon:

Answer 22

• Trp operon has 5 structural genes (TrpR binds at the operator, and the attenuator is a transcription terminator).

Question 23

There are two deletion mutations in the Trp operon that both have different effects, what are they?

Answer 23

• DeltaLD102: interferes with the operator, so an increase in expression is observed.
• DeltaED53: effects a different regulatory mechanism