BIO230 Lecture 3 Flashcards
Define: positive regulation
Activator protein recruits RNA Pol to the promotor to activate transcription
Define: negative regulation
Competition between RNA Pol and repressor protein for promoter binding
Define: inducing ligand
A ligand which when bound to a repressor protein removes the repressor protein and initiates transcription
Define: inhibitory ligand
A ligand which when bound to repressor protein switches the gene off
What are the 2 ways in which prokaryotic genes are negatively regulated?
- Gene off when repressor protein is bound to the operator; addition of ligand removes the repressor protein and initiates transcription
- Gene off when repressor protein is bound to the operator (with ligand); removal of ligand removes the repressor protein and turns gene on.
What are the 2 ways in which prokaryotic genes are positively regulated?
- Activator protein bound to RNA Pol (gene is on); addition of ligand removes the activator protein and turns gene off.
- Activator protein bound to RNA Pol with ligand (gene is on); removal of ligand removes activator protein and turns gene off.
Example: a negatively regulated prokaryotic gene that is activated when a ligand binds.
LAC repressor
Example: a negatively regulated prokaryotic gene that is activated when a ligand is removed.
Trp repressor
Example: a positively regulated prokaryotic gene that is activated when a ligand binds.
Catabolite Activator Protein (CAP)
Where are gene regulatory elements typically found in prokaryotes?
close to the transcriptional start site of prokaryotic genes
Where are some places that regulatory elements can be found (not the usual case) in both eukaryotes and prokaryotes?
- far upstream of the gene
- downstream of the gene (only eukaryotes)
- within the gene (in introns -> only eukaryotes)
How do regulatory elements that are far from the transcriptional start site influence transcription?
DNA looping
DNA looping _______ protein-DNA interactions
stabilizes
Describe DNA looping.
A repressor or activator (regulatory element) can bind to 2 operators simultaneously. Once the element binds to one operator (transition state), it is very likely to bind to second operator (double bond is energetically favourable). Once bound, it is very stable and tight.
Give an example of a regulatory element that uses DNA looping.
Lac repressor (tetramer) that can bind to 2 operators simultaneously.
What are the names of the two operators involved during DNA looping?
O_m (main operator)
O_a (auxiliary operator)
Define: bacteriophage lambda
A VIRUS that infects bacterial cells.
What is bacteriophage lambda’s genome like?
- Double stranded DNA
- 50000 nucleotide pairs
- Encodes 50-60 proteins
____ and _____ regulatory mechanisms work together to regulate the lifestyle of bacteriophage lambda. How?
Positive; negative
repress each other’s synthesis.
What is the anatomy of bacteriophage lambda?
(Red) DNA surrounded by (green) protein shell - has a protein tail!
Describe the process in which bacteriophage lambda gets into the bacterial host cell.
- lambda virus attaches to host cell and injects its DNA
2. lambda DNA circularizes (proteins aid the process)
What are the stages that bacteriophage lambda can exist in bacteria?
- Favourable bacterial growth conditions
2. Host cell damanged
Describe what happens when bacterial cell growth is favourable and bacteriophage lambda is inside?
- lambda DNA combines into host cell DNA
- integrated lambda DNA replicates along with host chromosome
Virus DNA hides in host cell DNA and gets replicated along with it.
Describe what happens when bacterial cell is damaged and bacteriophage lambda is inside?
- virus makes proteins needed for formation of new viruses
- proteins form protein shells
- replication of lambda DNA -> combined with protein shells and make complete viruses
- replicate until enough to cause bacteria cell to lyse
- large number of new viruses released -> look for new hosts
What is the pathway that bacteriophage lambda takes when the host cell growth is favourable? What if it’s unfavourable?
Favourable = prophage pathway Unfavourable = lytic pathway
What is responsible for the switch between the prophage pathway and the lytic pathway?
2 gene regulatory proteins:
1) Lambda repressor protein (cI) 2) Cro protein
What is the relationship between the lambda repressor protein (cI) and the Cro protein?
Repress each other’s synthesis to give rise to the two states (prophage/lytic)
In the stable state 1 (prophage state), what happens?
lambda repressor protein (cI) made.
- activate synthesis of itself -> help RNA Pol bind to make more of cI -> binds to operator (cycle)
- blocks synthesis of Cro
- most bacteriophage DNA not transcribed
In stable state 2 (lytic state), what happens?
lambda Cro protein is made
- Allows synthesis of more Cro protein -> allow RNA Pol to bind
- Blocks synthesis of lambda repressor protein
- Most bacteriophage DNA extensively transcribed
Which direction does RNA Pol go if cI is bound to the operator?
left
Which direction does RNA Pol go if Cro is bound to the operator?
Right
What triggers switch between prophage and lytic states?
- Host response to DNA damage will inactivates cI -> switch to lytic state -> Cro activated
- Good growth conditions: Ci turns off Cro -> activates self -> prophage state
What type of feedback loop does cI operate under?
positive feedback loop
Stimulus: good growth conditions for host cell
- Turns off Cro
- Activates self (cI)
- cI binds to operator
- More cI
Define: transcriptional circuit
Give an example.
Different gene regulatory proteins determine what is being transcribed (????)
Ex. Bacteriophage lambda -> switch between prophage and lytic states
What are 4 types of transcriptional circuits and explain each.
- Positive feedback loop: facilitate more transcription to make more of itself
- Negative feedback loop: repress transcription to stop production of itself
- Flip-flop device: A stops transcription of B; B stops transcription of A
- Feed-forward loop: Increase in A causes increase in B which will cause increase in Z. Increase in A will also cause increase in Z
What is cell memory and what can be used to create it?
Daughter cells (and all future cells) remember what the original parent cell got a external transient signal to turn on protein A, and therefore do not need the stimulus to turn on protein A.
What is one thing that cell memory is useful for?
Cell specialization.
Why are feed forward loops used?
Make the reaction to stimulus not too fast or slow.
- if only A regulates, then will react too rashly/fast
- if only A -> B -> C, then will be too slow
Allow cell to ignore rapid fluctuations in input signal.
Brief input will cause B to not accumulate, and Z will…
Prolonged input cause B to accumulate, and Z will…
- not be transcribed
- transcribed, but concave down positive slope (start when B signal is received until signal disappears), then concave up negative slope (after B has disappeared).
What is the shape of the B signal graph of a feed forward loop?
Straight up and down when signal is received/over.
Describe the shape of the Z graph when B receives prolonged signal (feed forward loop)
concave down positive slope (start when B signal is received until signal disappears), then concave up negative slope (after B has disappeared).
Combinations of regulatory circuits in eukaryotes create….
complex regulatory networks
Using _____, scientists can construct artificial regulatory circuits and examine their behaviour.
synthetic biology
What is an example of something created by synergetic biology?
the repressillator
What is the repressillator?
- Created by synthetic biology
- simple gene oscillator using delayed negative feedback circuit
How does the repressillator work?
3 genes:
A = Lac repressor
B = Tet repressor (response to antibiotic)
C = Lambda repressor
A transcribes genes -> bind to A binding site on B (represses B) -> nothing bind to C so C transcribes -> bind to C binding site on A -> A not produced -> nothing bound to B so B transcribes -> B bind to B binding site on C so C is not transcribed….etc.
For the repressillator, how does the predicted result differ from the observed?
Predicted: proteins A, B, C will fluctuate up and down in an oscillating cycle and the main graph will increase until it reaches a plateau.
Actual: similar, but the big graph will also oscillate up and down in an overall increasing direction because BACTERIAL MULTIPLIES AND GROWS
bacteria growth causes overall amount of gene to go up over time.
For the repressillator, what is the x and y axis of the graph?
x = time in minutes
y = proteins per cell (predicted)
= fluorescence of one gene (observed)
An example of feedback loop is circadian gene regulation. Give an example and describe it.
Drosophila circadian cycle:
- Tim (timeless) and Per (period) genes are both encoded by mRNA and form a diamer
> Tim controls night time functions (degrades in response to light)
> Per controls daytime functions, phosphorylated and degraded
- when enough is produced the diamer dissociates and goes back into nucleus
- Per degrades Per and Tim to prevent periodic accumulation
Define: transcription attenuation
Premature termination of transcription in both prokaryotes and eukaryotes
How does transcription attenuation work?
- RNA adopt structure that interferes with RNA Pol
- Regulatory proteins bind to RNA & interfere with attenuation
What do prokaryotes, plants, and some fungi use to regulate gene expression?
Riboswitches
Define: riboswitches
Short RNA sequences that change conformations when bound by a small molecule
Example: riboswitch
Prokaryotic riboswitch that regulates purine biosynthesis
How does prokaryotic riboswitch work when regulating purine biosynthesis?
Low guanine levels turn transcription of purine biosynthetic genes on (enzymes needed for guanine synthesis is expressed)
High guanine levels: guanine binds to riboswitch -> riboswitch undergoes conformational change -> RNA polymerase terminates transcription -> transcription of purine biosynthetic gene is off
